Anti-tigit antibodies

ABSTRACT

The present invention relates to anti-TIGIT antibodies, as well as use of these antibodies in the treatment of diseases such as cancer and infectious disease.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 27, 2015, isnamed 23808-US-PCT_SL.txt and is 136,039 bytes in size.

FIELD OF THE INVENTION

The present invention relates to anti-TIGIT antibodies, as well as useof these antibodies in the treatment of diseases such as cancer andinfectious disease.

BACKGROUND OF THE INVENTION

A key factor for enabling tumor immunotherapy emerged from discoveriesthat inhibitory immune modulatory receptors (IMRs), that generallyfunction as immune checkpoints to maintain self-tolerance, are centralto the ability of tumor microenvironments to evade immunity. Blockade ofinhibitory IMRs appears to unleash potent tumor-specific immuneresponses more effectively than direct stimulation of tumor-immunitywith activating cytokines or tumor vaccines, and this approach has thepotential to transform human cancer therapy. An important implicationand opportunity now arises for the potential to develop new antibodyantagonists for other IMRs and to combine antagonist antibodies to morethan one IMR in order to increase the proportion of responders inoncology clinical trials, as well as, expand upon oncology indicationsin which tumor immunotherapy treatments are effective.

Significantly, inhibitory IMRs and ligands that regulate cellularimmunity are commonly overexpressed on tumor cells and tumor associatedmacrophages (TAMs). Notably, overexpression of PD-L1 in tumors isassociated with tumor specific T cell exhaustion and a poor prognosis.Blockade of PD-1/PD-L1 ligation in clinical trials resulted in durabletumor regression responses in a substantial proportion of patients. Arecent report demonstrated that co-expression of PD-1 and anotherinhibitory IMR (TIM-3) in melanoma patient derived tumor-specific CD8+ Tcells was associated with more dysfunctional T cell exhaustionphenotypes compared to cells expressing either IMR alone. Moreover,several reports using pre-clinical tumor models demonstrated blockade ofmultiple IMRs, including PD-1, TIM-3, LAG-3 and CTLA-4 more effectivelyinduced anti-tumor responses than antagonizing PD-1 alone. These resultsunderscore the importance of further investigating IMR pathways.

TIGIT (T cell immunoreceptor with Ig and ITIM domains) is animmunomodulatory receptor expressed primarily on activated T cells andNK cells. TIGIT is also known as VSIG9; VSTM3; and WUCAM. Its structureshows one extracellular immunoglobulin domain, a type 1 transmembraneregion and two ITIM motifs. TIGIT forms part of a co-stimulatory networkthat consists of positive (CD226) and negative (TIGIT) immunomodulatoryreceptors on T cells, and ligands expressed on APCs (CD155 and CD112).

An important feature in the structure of TIGIT is the presence of animmunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmictail domain. As with PD-1 and CTLA-4, the ITIM domain in the cytoplasmicregion of TIGIT is predicted to recruit tyrosine phosphatases, such asSHP-1 and SHP-2, and subsequent de-phosphorylation of tyrosine residueswith in the immunoreceptor tyrosine-base activation motifs (ITAM) on Tcell receptor (TCR) subunits. Hence, ligation of TIGIT byreceptor-ligands CD155 and CD112 expressed by tumor cells or TAMS maycontribute to the suppression of TCR-signaling and T cell activation,which is essential for mounting effective anti-tumor immunity. Thus, anantagonist antibody specific for TIGIT could inhibit the CD155 and CD112induced suppression of T cell responses and enhance anti-tumor immunity.It is an object of the present invention to obtain an anti-TIGITantibody that can be used for the treatment of cancer, either alone orin combination with other reagents.

SUMMARY OF THE INVENTION

The invention provides anti-TIGIT antibodies and antigen bindingfragments thereof comprising the structural and functional featuresspecified below.

In one embodiment, the invention provides an antibody or antigen bindingfragment thereof that binds to human TIGIT, comprising: a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3,79, 80, 81, 82, 83, 59, 90, 140, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166 or 167. In one embodiment, the antibody orantigen binding fragment thereof optionally has at least one of thefollowing characteristics: (i) binds to human TIGIT with a KD value ofabout 1×10⁻⁹M to about 1×10⁻¹² M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e g KinExa or OCTET);(ii) cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocksbinding of human TIGIT to human CD155 and human CD112; (iv) increases Tcell activation; (v) stimulates antigen-specific T-cell production ofIL-2 and IFNγ; (vi) blocks induction of T cell suppression of activationinduced by TIGIT ligation with cognate ligands CD155 and CD112.

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that binds to human TIGIT, comprising: a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 6, 62, 74, 75, 76, 77, 78 or 93. In one embodiment, the antibodyoptionally has at least one of the following characteristics: (i) bindsto human TIGIT with a KD value of about 1×10⁻⁹ M to about 1×10⁻¹² M asdetermined by surface plasmon resonance (e.g., BIACORE) or a similartechnique (e.g. KinExa or OCTET); (ii) cross-reacts with cynomolgous andrhesus TIGIT; (iii) blocks binding of human TIGIT to human CD155 andhuman CD112; (iv) increases T cell activation; (v) stimulatesantigen-specific T-cell production of IL-2 and IFNγ; (vi) blocksinduction of T cell suppression of activation induced by TIGIT ligationwith cognate ligands CD155 and CD112.

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that binds to human TIGIT comprising: (i) aheavy chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 2; and (iii) a heavy chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 140. In oneembodiment, the antibody or antigen binding fragment comprises: (i) aheavy chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 2; and (iii) a heavy chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 79, 80,81, 82 or 83. In one embodiment, the antibody or antigen bindingfragment thereof comprises: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2;and (iii) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 3. In one embodiment, the antibody or antigenbinding fragment thereof optionally has at least one of the followingcharacteristics: (i) binds to human TIGIT with a KD value of about1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmon resonance(e.g., BIACORE) or a similar technique (e g KinExa or OCTET); (ii)cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocks binding ofhuman TIGIT to human CD155 and human CD112; (iv) increases T cellactivation; (v) stimulates antigen-specific T-cell production of IL-2and IFNγ; (vi) blocks induction of T cell suppression of activationinduced by TIGIT ligation with cognate ligands CD155 and CD112.

In another embodiment, the invention provides an antibody or antigenbinding fragment that binds to human TIGIT comprising: (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:57; (ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 58; and (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 59. In one embodiment,the antibody or antigen binding fragment thereof optionally has at leastone of the following characteristics: (i) binds to human TIGIT with a KDvalue of about 1×10⁻⁹M to about 1×10⁻¹² M as determined by surfaceplasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa orOCTET); (ii) cross-reacts with cynomolgous and rhesus TIGIT; (iii)blocks binding of human TIGIT to human CD155 and human CD112; (iv)increases T cell activation; (v) stimulates antigen-specific T-cellproduction of IL-2 and IFNγ; (vi) blocks induction of T cell suppressionof activation induced by TIGIT ligation with cognate ligands CD155 andCD112.

In another embodiment, the invention provides an antibody or antigenbinding fragment that binds to human TIGIT comprising: (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:88; (ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 147; and (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 153. In one embodiment,the antibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 89, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 134 or 135; and (iii) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO:90, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 or167. In one embodiment, the antibody or antigen binding fragment thereofcomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 89, 134 or 135; and(iii) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 90. In one embodiment, the antibody or antigenbinding fragment thereof comprises: (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavychain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 134; and (iii) a heavy chain variable region CDR3 comprising theamino acid sequence of SEQ ID NO: 90. In one embodiment, the antibody orantigen binding fragment thereof comprises: (i) a heavy chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 88; (ii) aheavy chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 134; and (iii) a heavy chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 154. In one embodiment, theantibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 134; and (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 155. In oneembodiment, the antibody or antigen binding fragment thereof comprises:(i) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; and (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 156. In one embodiment, the antibody or antigen binding fragmentthereof comprises: (i) a heavy chain variable region CDR1 comprising theamino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable regionCDR2 comprising the amino acid sequence of SEQ ID NO: 134; and (iii) aheavy chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 157. In one embodiment, the antibody or antigen bindingfragment thereof comprises: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:134; and (iii) a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 158. In one embodiment, the antibody orantigen binding fragment thereof comprises: (i) a heavy chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 88; (ii) aheavy chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 134; and (iii) a heavy chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 159. In one embodiment, theantibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 134; and (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 160. In oneembodiment, the antibody or antigen binding fragment thereof comprises:(i) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; and (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 161. In one embodiment, the antibody or antigen binding fragmentthereof comprises: (i) a heavy chain variable region CDR1 comprising theamino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable regionCDR2 comprising the amino acid sequence of SEQ ID NO: 134; and (iii) aheavy chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 162. In one embodiment, the antibody or antigen bindingfragment thereof comprises: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:134; and (iii) a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 163. In one embodiment, the antibody orantigen binding fragment thereof comprises: (i) a heavy chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 88; (ii) aheavy chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 134; and (iii) a heavy chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 164. In one embodiment, theantibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 134; and (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 165. In oneembodiment, the antibody or antigen binding fragment thereof comprises:(i) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; and (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 166. In one embodiment, the antibody or antigen binding fragmentthereof comprises: (i) a heavy chain variable region CDR1 comprising theamino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable regionCDR2 comprising the amino acid sequence of SEQ ID NO: 134; and (iii) aheavy chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 167. In one embodiment, the antibody or antigen bindingfragment thereof optionally has at least one of the followingcharacteristics: (i) binds to human TIGIT with a KD value of about1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmon resonance(e.g., BIACORE) or a similar technique (e g KinExa or OCTET); (ii)cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocks binding ofhuman TIGIT to human CD155 and human CD112; (iv) increases T cellactivation; (v) stimulates antigen-specific T-cell production of IL-2and IFNγ; (vi) blocks induction of T cell suppression of activationinduced by TIGIT ligation with cognate ligands CD155 and CD112.

In another embodiment, the invention provides an antibody or antigenbinding fragment that binds to human TIGIT comprising: (i) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4;(ii) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 141; and (iii) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 142. In one embodiment,the antibody or antigen binding fragment thereof comprises: (i) a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 4; (ii) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 5, 65, 66, 67, 68, 69, 70, 71, 72 or; and (iii) alight chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 6, 74, 75, 76, 77 or 78. In one embodiment, the antibody orantigen binding fragment thereof comprises: (i) a light chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) alight chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 5; and (iii) a light chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 6. In one embodiment, the antibodyor antigen binding fragment thereof optionally has at least one of thefollowing characteristics: (i) binds to human TIGIT with a KD value ofabout 1×10⁻⁹M to about 1×10⁻¹² M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e g KinExa or OCTET);(ii) cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocksbinding of human TIGIT to human CD155 and human CD112; (iv) increases Tcell activation; (v) stimulates antigen-specific T-cell production ofIL-2 and IFNγ; (vi) blocks induction of T cell suppression of activationinduced by TIGIT ligation with cognate ligands CD155 and CD112.

In another embodiment, the invention provides an antibody or antigenbinding fragment that binds to human TIGIT comprising: (i) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:60; (ii) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 61; and (iii) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 62. In one embodiment,the antibody or antigen binding fragment thereof optionally has at leastone of the following characteristics: (i) binds to human TIGIT with a KDvalue of about 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surfaceplasmon resonance (e.g., BIACORE) or a similar technique (e g KinExa orOCTET); (ii) cross-reacts with cynomolgous and rhesus TIGIT; (iii)blocks binding of human TIGIT to human CD155 and human CD112; (iv)increases T cell activation; (v) stimulates antigen-specific T-cellproduction of IL-2 and IFNγ; (vi) blocks induction of T cell suppressionof activation induced by TIGIT ligation with cognate ligands CD155 andCD112.

In another embodiment, the invention provides an antibody or antigenbinding fragment that binds to human TIGIT comprising: (i) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:91; (ii) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 148; and (iii) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 93. In one embodiment,the antibody or antigen binding fragment thereof comprises: (i) a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 91; (ii) a light chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 92, 112, 113, 114, 115, 116, 117, 118, 119,120, 121 or 122; and (iii) a light chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 93. In one embodiment, theantibody or antigen binding fragment thereof comprises: (i) a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 91; (ii) a light chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 92; and (iii) a light chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 93. In oneembodiment, the antibody or antigen binding fragment thereof optionallyhas at least one of the following characteristics: (i) binds to humanTIGIT with a KD value of about 1×10⁻⁹ M to about 1×10⁻¹² M as determinedby surface plasmon resonance (e.g., BIACORE) or a similar technique (e gKinExa or OCTET); (ii) cross-reacts with cynomolgous and rhesus TIGIT;(iii) blocks binding of human TIGIT to human CD155 and human CD112; (iv)increases T cell activation; (v) stimulates antigen-specific T-cellproduction of IL-2 and IFNγ; (vi) blocks induction of T cell suppressionof activation induced by TIGIT ligation with cognate ligands CD155 andCD112.

In another embodiment, the invention provides an antibody or antigenbinding fragment that binds to human TIGIT comprising: (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1;(ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 140; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4;(v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 141; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 142. In one embodiment,the antibody or antigen binding fragment comprising: (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1;(ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 3, 79, 80, 81, 82 or83; (iv) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5, 65, 66, 67, 68, 69,70, 71, 72 or 73; and (vi) a light chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 6, 74, 75, 76, 77 or 78. In oneembodiment, the antibody or antigen binding fragment thereof comprises:(i) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3;(iv) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 6. In one embodiment, the antibody or antigen binding fragmentthereof is humanized. In one embodiment, the antibody or antigen bindingfragment thereof comprises a heavy chain variable region selected fromthe group consisting of SEQ ID NOs:9-24, 37-47, 143 and 144; and a lightchain variable region selected from the group consisting of SEQ ID NOs:25-30, 48-52, 146 and 147. In one embodiment, the antibody or antigenbinding fragment thereof optionally has at least one of the followingcharacteristics: (i) binds to human TIGIT with a KD value of about1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmon resonance(e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); (ii)cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocks binding ofhuman TIGIT to human CD155 and human CD112; (iv) increases T cellactivation; (v) stimulates antigen-specific T-cell production of IL-2and IFNγ; (vi) blocks induction of T cell suppression of activationinduced by TIGIT ligation with cognate ligands CD155 and CD112.

In another embodiment, the invention provides an antibody or antigenbinding fragment that binds to human TIGIT comprising: (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1;(ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 3, 79, 80, 81, 82 or83; (iv) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5, 65, 66, 67, 68, 69,70, 71, 72 or 73; and (vi) a light chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 6, 74, 75, 76, 77 or 78; whereinthe antibody or antigen binding fragment thereof comprises a heavy chainvariable region comprising at least 90%, 95%, 96%, 97%, 98% or 99%identity to a heavy chain variable region selected from the groupconsisting of SEQ ID NOs: 9-24 or 37-47 and a light chain variableregion comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity to aheavy chain variable region selected from the group consisting of SEQ IDNOs: 25-30 or 48-52. In this aforementioned embodiment, the sequencevariations occur in the framework regions. In one embodiment, theantibody binds to human TIGIT with a KD value of about 1×10⁻⁹M to about1×10⁻¹² M as determined by surface plasmon resonance (e.g., BIACORE) ora similar technique (e.g. KinExa or OCTET).

In another embodiment, the invention also provides an antibody orantigen binding fragment thereof that binds to human TIGIT comprising:(i) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3;(iv) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 6. In one embodiment, the antibody or antigen binding fragmentthereof comprises 1, 2 or 3 amino acid substitutions in the heavy chainCDRs (SEQ ID NOs: 1-3) or in the light chain CDRs (SEQ ID NOs: 3-6). Inone embodiment, the antibody comprises one amino acid substitution inthe heavy chain CDR of SEQ ID NO: 3, wherein the substitution is made atposition 13W, and wherein residue 13W is substituted to: F, Y, I, V orL. In one embodiment, the antibody comprises two amino acidsubstitutions in the light chain CDR of SEQ ID NO: 5, wherein thesubstitutions are made at position 3-4, and wherein residues 3N and 4Sare substituted to: SN, SS, ST, TT, SY, NQ, GS, SQ and DS. In oneembodiment, the antibody comprises one amino acid substitution in thelight chain CDR of SEQ ID NO: 6, wherein the substitution is made atposition 7, and wherein residue 7W is substituted to: F, Y, I, V or L.The VH sequences of SEQ ID NOs: 9-24 and 37-47 have the CDRs of SEQ IDNOs:1-3; and VL sequences of SEQ ID NOs:25-30 and 48-52 have the CDRs ofSEQ ID NOs: 4-6. In some embodiments, the CDR substitutions describedabove can be made in the corresponding CDRs of the VH sequences of SEQID NOs: 9-24 and 37-37, and in the CDRs of the VL sequences of SEQ IDNOs: 25-30 and 48-52. In one embodiment, the antibody binds to humanTIGIT with a KD value of about 1×10⁻⁹M to about 1×10⁻¹² M as determinedby surface plasmon resonance (e.g., BIACORE) or a similar technique (e gKinExa or OCTET).

In another embodiment, the invention provides an antibody or antigenbinding fragment that binds to human TIGIT comprising: (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:57; (ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 58; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 59; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:60; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 61; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 62. In one embodiment,the antibody or antigen binding fragment thereof is humanized. In oneembodiment, the antibody or antigen binding fragment thereof comprises1, 2 or 3 amino acid substitutions in the heavy chain CDRs (SEQ ID NOs:57-59) or in the light chain CDRs (SEQ ID NOs: 60-62), and retains oneor more of its functional characteristics. In one embodiment, theantibody or antigen binding fragment thereof optionally has at least oneof the following functional characteristics: (i) binds to human TIGITwith a KD value of about 1×10⁻⁹ M to about 1×10⁻¹² M as determined bysurface plasmon resonance (e.g., BIACORE) or a similar technique (e gKinExa or OCTET), (ii) cross-reacts with cynomolgous and rhesus TIGIT;(iii) blocks binding of human TIGIT to human CD155 and human CD112; (iv)increases T cell activation; (v) stimulates antigen-specific T-cellproduction of IL-2 and IFNγ; (vi) blocks induction of T cell suppressionof activation induced by TIGIT ligation with cognate ligands CD155 andCD112.

In another embodiment, the invention also provides an antibody orantigen binding fragment thereof that binds to human TIGIT comprising:(i) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 147; (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 153; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 148; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 93. In one embodiment, the antibody or antigen binding fragmentthereof comprises: (i) a heavy chain variable region CDR1 comprising theamino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable regionCDR2 comprising the amino acid sequence of SEQ ID NO: 89, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 134 or135; (iii) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 90, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166 or 167; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 91; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:92, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122; and (vi) alight chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 93. In one embodiment, the antibody or antigen bindingfragment thereof comprises: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:89, 134 or 135; (iii) a heavy chain variable region CDR3 comprising theamino acid sequence of SEQ ID NO: 90; (iv) a light chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 91; (v) a lightchain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 92; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 93. In one embodiment, the antibody orantigen binding fragment thereof comprises: (i) a heavy chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 88; (ii) aheavy chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 134; (iii) a heavy chain variable region CDR3 comprising theamino acid sequence of SEQ ID NO: 90; (iv) a light chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 91; (v) a lightchain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 92; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 93. In one embodiment, the antibody orantigen binding fragment thereof is humanized. In one embodiment, theantibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 134; (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 154; (iv) a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 91; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 92; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 93. In one embodiment,the antibody or antigen binding fragment thereof is humanized. In oneembodiment, the antibody or antigen binding fragment thereof comprises:(i) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 155; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 92; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 93. In one embodiment, the antibody or antigen binding fragmentthereof is humanized. In one embodiment, the antibody or antigen bindingfragment thereof comprises: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:134; (iii) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 156; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 91; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:92; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 93. In one embodiment, the antibody orantigen binding fragment thereof is humanized. In one embodiment, theantibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 134; (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 157; (iv) a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 91; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 92; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 93. In one embodiment,the antibody or antigen binding fragment thereof is humanized. In oneembodiment, the antibody or antigen binding fragment thereof comprises:(i) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 158; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 92; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 93. In one embodiment, the antibody or antigen binding fragmentthereof is humanized. In one embodiment, the antibody or antigen bindingfragment thereof comprises: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:134; (iii) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 159; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 91; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:92; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 93. In one embodiment, the antibody orantigen binding fragment thereof is humanized. In one embodiment, theantibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 134; (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 160; (iv) a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 91; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 92; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 93. In one embodiment,the antibody or antigen binding fragment thereof is humanized. In oneembodiment, the antibody or antigen binding fragment thereof comprises:(i) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 161; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 92; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 93. In one embodiment, the antibody or antigen binding fragmentthereof is humanized. In one embodiment, the antibody or antigen bindingfragment thereof comprises: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:134; (iii) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 162; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 91; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:92; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 93. In one embodiment, the antibody orantigen binding fragment thereof is humanized. In one embodiment, theantibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 134; (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 163; (iv) a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 91; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 92; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 93. In one embodiment,the antibody or antigen binding fragment thereof is humanized. In oneembodiment, the antibody or antigen binding fragment thereof comprises:(i) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 164; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 92; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 93. In one embodiment, the antibody or antigen binding fragmentthereof is humanized. In one embodiment, the antibody or antigen bindingfragment thereof comprises: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:134; (iii) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 165; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 91; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:92; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 93. In one embodiment, the antibody orantigen binding fragment thereof is humanized. In one embodiment, theantibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 134; (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 166; (iv) a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 91; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 92; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 93. In one embodiment,the antibody or antigen binding fragment thereof is humanized. In oneembodiment, the antibody or antigen binding fragment thereof comprises:(i) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 167; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 92; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 93. In one embodiment, the antibody or antigen binding fragmentthereof is humanized. In one embodiment, the antibody or antigen bindingfragment thereof optionally has at least one of the followingcharacteristics: (i) binds to human TIGIT with a KD value of about1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmon resonance(e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET), (ii)cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocks binding ofhuman TIGIT to human CD155 and human CD112; (iv) increases T cellactivation; (v) stimulates antigen-specific T-cell production of IL-2and IFNγ; (vi) blocks induction of T cell suppression of activationinduced by TIGIT ligation with cognate ligands CD155 and CD112.

In one embodiment, the antibody or antigen binding fragment thereofbinds to human TIGIT and comprises: (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavychain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 89; (iii) a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 90; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 91; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:92; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 93. In one embodiment, the antibodycomprises one amino acid substitution in the heavy chain CDR2 of SEQ IDNO: 89, wherein the substitution is made at position 7, and whereinresidue D is substituted with: R, L, K, F, S, Y or V. In anotherembodiment, the antibody comprises one amino acid substitution in theheavy chain CDR2 of SEQ ID NO: 89, wherein the substitution is made atposition 8, and wherein residue G is substituted with: R, N, Q, E, L K,S, Y or V. In another embodiment, the antibody comprises one amino acidsubstitution in the heavy chain CDR2 of SEQ ID NO: 89, wherein thesubstitution is made at position 12, wherein the N is substituted with:A or S. In another embodiment, the antibody comprises one amino acidsubstitution in the heavy chain CDR2 of SEQ ID NO: 89, wherein thesubstitution is made at position 13, wherein E is substituted with Q. Inanother embodiment, the antibody comprises one amino acid substitutionin the heavy chain CDR2 of SEQ ID NO: 89, wherein the substitution ismade at position 16, wherein K is substituted with Q. In anotherembodiment, the antibody comprises three amino acid substitution in theheavy chain CDR2 of SEQ ID NO: 89, wherein amino acid residue 12 issubstituted with: A or S, amino acid residue 13 is substituted with Qand amino acid residue 16 is substituted with Q. In one embodiment, theantibody comprises one amino acid substitution in the heavy chain CDR3of SEQ ID NO:90, wherein amino acid residue 6 is substituted with: A, D,E, F, G, I, K, N, Q, R, S, T, V or Y. In another embodiment, theantibody comprises one amino acid substitution in the light chain CDR ofSEQ ID NO: 92, wherein the substitution is made at position 1, andwherein residue N is substituted with A, Y, W, S, T, R, H, G, I or V. Inanother embodiment, the antibody comprises one amino acid substitutionin the light chain CDR of SEQ ID NO: 92, wherein the substitution ismade at position 2, and wherein residue A is substituted with N, I, L,T, V. In one embodiment, the antibody or antigen binding fragmentthereof is humanized. In one embodiment, the antibody or antigen bindingfragment thereof optionally has at least one of the followingcharacteristics: (i) binds to human TIGIT with a KD value of about1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmon resonance(e.g., BIACORE) or a similar technique (e g KinExa or OCTET), (ii)cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocks binding ofhuman TIGIT to human CD155 and human CD112; (iv) increases T cellactivation; (v) stimulates antigen-specific T-cell production of IL-2and IFNγ; (vi) blocks induction of T cell suppression of activationinduced by TIGIT ligation with cognate ligands CD155 and CD112.

In another embodiment, the invention also provides an antibody orantigen binding fragment thereof that binds to human TIGIT comprising: aheavy chain variable region comprising the CDR1, CDR2 and CDR3 of any ofSEQ ID NOs: 124-129 and/or a light chain variable region comprising theCDR1, CDR2 and CDR3 of any of SEQ ID NOs:130-133. In one embodiment, theinvention also provides an isolated antibody or antigen binding fragmentthereof that binds to human TIGIT comprising: a heavy chain variableregion selected from the group consisting of: SEQ ID NOs: 124-129 and/ora light chain variable region selected from the group consisting of: SEQID NOs: 130-133. In one embodiment, the D residue at position 56 of anyone of SEQ ID NOs: 124-129 can be substituted with R, L, K, F, S, Y orV. In another embodiment, the G residue at position 57 of any one of SEQID NOs: 124-129 can be substituted with R, N, Q, E, L K, S, Y or V. Inone embodiment, the W residue at position 104 of any one of SEQ ID NOs:124-129 can be substituted with: A, D, E, F, G, I, K, N, Q, R, S, T, Vor Y. In another embodiment, the N residue at position 50 of any one ofSEQ ID NOs: 130-133 can be substituted with A, Y, W, S, T, I or V. Inanother embodiment, the A residue at position 51 of any of SEQ ID NOs:130-133 is substituted with N, I, L, T or V. In one embodiment, theinvention provides an isolated antibody or antigen binding fragmentthereof that binds to human TIGIT comprising: a heavy chain variableregion comprising SEQ ID NO:128 or a light chain variable regioncomprising SEQ ID NO:132. In another embodiment, the invention providesan isolated antibody or antigen binding fragment thereof that binds tohuman TIGIT comprising: a heavy chain variable region comprising SEQ IDNO:128 or a light chain variable region comprising SEQ ID NO:133. Inanother embodiment, the invention also provides an isolated antibody orantigen binding fragment thereof that binds to human TIGIT comprising: aheavy chain variable region comprising SEQ ID NO:127 or a light chainvariable region comprising SEQ ID NO:130. In one embodiment, theinvention provides an isolated antibody or antigen binding fragmentthereof that binds to human TIGIT comprising: a heavy chain variableregion comprising SEQ ID NO:128 and a light chain variable regioncomprising SEQ ID NO:132. In another embodiment, the invention alsoprovides an isolated antibody or antigen binding fragment thereof thatbinds to human TIGIT comprising: a heavy chain variable regioncomprising SEQ ID NO:128 and a light chain variable region comprisingSEQ ID NO:133. In another embodiment, the invention also provides anisolated antibody or antigen binding fragment thereof that binds tohuman TIGIT comprising: a heavy chain variable region comprising SEQ IDNO:126 and a light chain variable region comprising SEQ ID NO:131. Inanother embodiment, the invention also provides an isolated antibody orantigen binding fragment thereof that binds to human TIGIT comprising: aheavy chain variable region comprising SEQ ID NO:128 and a light chainvariable region comprising SEQ ID NO:131. In another embodiment, theinvention also provides an isolated antibody or antigen binding fragmentthereof that binds to human TIGIT comprising: a heavy chain variableregion comprising SEQ ID NO:125 and a light chain variable regioncomprising SEQ ID NO:133. In another embodiment, the invention alsoprovides an isolated antibody or antigen binding fragment thereof thatbinds to human TIGIT comprising: a heavy chain variable regioncomprising SEQ ID NO:126 and a light chain variable region comprisingSEQ ID NO:130. In another embodiment, the invention also provides anisolated antibody or antigen binding fragment thereof that binds tohuman TIGIT comprising: a heavy chain variable region comprising SEQ IDNO:125 and a light chain variable region comprising SEQ ID NO:132. Inanother embodiment, the invention also provides an isolated antibody orantigen binding fragment thereof that binds to human TIGIT comprising: aheavy chain variable region comprising SEQ ID NO:127 and a light chainvariable region comprising SEQ ID NO:130. In some embodiments, theantibody or antigen binding fragment thereof binds to human TIGIT with aKD value of about 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surfaceplasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa orOCTET).

In another embodiment, the invention also provides an antibody orantigen binding fragment thereof that binds to human TIGIT comprising:(i) a heavy chain variable region comprising the CDR1, CDR2 and CDR3 ofany one of SEQ ID NOs: 124-129, wherein the heavy chain variable regioncomprises at least 90%, 95%, 96%, 97%, 98% or 99% identity to any of oneSEQ ID NOs: 124-129 and/or (ii) a light chain variable region comprisingthe CDR1, CDR2 and CDR3 of any one of SEQ ID NOs: 130-133, wherein thelight chain variable region comprises at least 90%, 95%, 96%, 97%, 98%or 99% identity to any one of SEQ ID NOs: 130-133. In anotherembodiment, the invention also provides an antibody or antigen bindingfragment thereof that binds to human TIGIT comprising: (i) a heavy chainvariable region comprising the CDR1, CDR2 and CDR3 of SEQ ID NOs: 128,wherein the heavy chain variable region comprises 90%, 95%, 96%, 97%,98% or 99% identity to SEQ ID NOs: 128 and/or (ii) a light chainvariable region comprising the CDR1, CDR2 and CDR3 of SEQ ID NOs: 132,wherein the light chain variable region comprises at least 90%, 95%,96%, 97%, 98% or 99% identity to SEQ ID NOs: 132. In another embodiment,the invention also provides an antibody or antigen binding fragmentthereof that binds to human TIGIT comprising: (i) a heavy chain variableregion comprising the CDR1, CDR2 and CDR3 of SEQ ID NOs: 128, whereinthe heavy chain variable region comprises at least 90%, 95%, 96%, 97%,98% or 99% identity to SEQ ID NOs: 128 and/or (ii) a light chainvariable region comprising the CDR1, CDR2 and CDR3 of SEQ ID NOs: 133,wherein the light chain variable region comprises at least 90%, 95%,96%, 97%, 98% or 99% identity to SEQ ID NOs: 133. In another embodiment,the invention also provides an antibody or antigen binding fragmentthereof that binds to human TIGIT comprising: (i) a heavy chain variableregion comprising the CDR1, CDR2 and CDR3 of SEQ ID NOs: 127, whereinthe heavy chain variable region comprises at least 90%, 95%, 96%, 97%,98% or 99% identity to SEQ ID NOs: 127 and/or (ii) a light chainvariable region comprising the CDR1, CDR2 and CDR3 of SEQ ID NOs: 130,wherein the light chain variable region comprises at least 90%, 95%,96%, 97%, 98% or 99% identity to SEQ ID NOs: 130. In these embodiments,the allowed sequence variations occur in the framework regions of thevariable chains. In some embodiments, the antibody or antigen bindingfragment thereof binds to human TIGIT with a KD value of about 1×10⁻⁹ Mto about 1×10⁻¹² M as determined by surface plasmon resonance (e.g.,BIACORE) or a similar technique (e.g. KinExa or OCTET).

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof, comprising: a variable heavy chain comprisingthe amino acid sequence of SEQ ID NO: 7 and/or a variable light chainselected comprising the amino acid sequence of SEQ ID NO: 8, wherein theantibody or antigen binding fragment thereof binds to human TIGIT. Inone embodiment, the antibody binds to human TIGIT with a KD value ofabout 1×10⁻⁹M to about 1×10⁻¹² M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET).

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof, comprising: a variable heavy chain comprisingthe amino acid sequence of SEQ ID NO: 143 and/or a variable light chainselected comprising the amino acid sequence of SEQ ID NO: 145, whereinthe antibody or antigen binding fragment thereof binds to human TIGIT.In one embodiment, the antibody binds to human TIGIT with a KD value ofabout 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET).

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof, comprising: a variable heavy chain comprisingthe amino acid sequence of SEQ ID NO: 144 and/or a variable light chainselected comprising the amino acid sequence of SEQ ID NO: 146, whereinthe antibody or antigen binding fragment thereof binds to human TIGIT.In one embodiment, the antibody binds to human TIGIT with a KD value ofabout 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET).

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof, comprising: a variable heavy chain comprisingthe amino acid sequence of SEQ ID NO: 63 and/or a variable light chainselected comprising the amino acid sequence of SEQ ID NO: 64, whereinthe antibody or antigen binding fragment thereof binds to human TIGIT.In one embodiment, the antibody binds to human TIGIT with a KD value ofabout 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET).

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof, comprising: a variable heavy chain comprisingthe amino acid sequence of SEQ ID NO: 94 and/or a variable light chainselected comprising the amino acid sequence of SEQ ID NO: 95, whereinthe antibody or antigen binding fragment thereof binds to human TIGIT.In one embodiment, the antibody binds to human TIGIT with a KD value ofabout 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET).

In one embodiment, the invention also provides an isolated antibody orantigen binding fragment thereof that binds to human TIGIT comprising: aheavy chain variable region comprising SEQ ID NO:149 and/or a lightchain variable region comprising SEQ ID NO:151. In one embodiment, theantibody binds to human TIGIT with a KD value of about 1×10⁻⁹ M to about1×10⁻¹² M as determined by surface plasmon resonance (e.g., BIACORE) ora similar technique (e.g. KinExa or OCTET).

In one embodiment, the invention also provides an isolated antibody orantigen binding fragment thereof that binds to human TIGIT comprising: aheavy chain variable region comprising SEQ ID NO:150 and/or a lightchain variable region comprising SEQ ID NO:152. In one embodiment, theantibody binds to human TIGIT with a KD value of about 1×10⁻⁹ M to about1×10⁻¹² M as determined by surface plasmon resonance (e.g., BIACORE) ora similar technique (e.g. KinExa or OCTET).

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that binds to the same epitope of human TIGITas an antibody comprising the variable heavy chain of SEQ ID NO: 7 andthe variable light chain of SEQ ID NO: 8, wherein the antibody orantigen binding fragment thereof has at least one of the followingcharacteristics: (i) binds to human TIGIT with a KD value of about1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmon resonance(e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET), (ii)blocks binding of human TIGIT to human CD155 and human CD112; (iii)increases T cell activation; (iv) stimulates antigen-specific T-cellproduction of IL-2 and IFNγ; (v) blocks induction of T cell suppressionof activation induced by TIGIT ligation with cognate ligands CD155 andCD112. In one embodiment, the antibody comprises at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity with the variable heavychain and/or the variable light chain of any one of SEQ ID NOs: 7-30 or37-52. In another embodiment, the antibody or antigen binding fragmentthereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acidsubstitutions in the variable heavy light chain of any one of SEQ IDNOs: 7, 9-24 and 37-47 and/or the variable light chains of any one ofSEQ ID NOs: 8, 25-30 and 48-52.

In one embodiment, the invention provides an antibody or antigen bindingfragment thereof that binds to an epitope of human TIGIT that comprisesat least one of the following regions: residues 54-57 of SEQ ID NO: 31,residues 68-70 of SEQ ID NO:31 and residues 76-81 of SEQ ID NO: 31. Inone embodiment, the invention provides an antibody or antigen bindingfragment thereof that binds to an epitope of human TIGIT comprisingresidues: 54-57, 68-70 and 76-81 of SEQ ID NO:31.

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that binds to the same epitope of human TIGITas an antibody comprising the variable heavy chain of SEQ ID NO: 63 andthe variable light chain of SEQ ID NO: 64, wherein the antibody orantigen binding fragment thereof has at least one of the followingcharacteristics: (i) binds to human TIGIT with a KD value of about1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmon resonance(e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); (ii)cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocks binding ofhuman TIGIT to human CD155 and human CD112; (iv) increases T cellactivation; (v) stimulates antigen-specific T-cell production of IL-2and IFNγ; (vi) blocks induction of T cell suppression of activationinduced by TIGIT ligation with cognate ligands CD155 and CD112. In oneembodiment, the antibody or antigen binding fragment thereof comprisesat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity withthe variable heavy chain of SEQ ID NO:63 and/or the variable light chainof SEQ ID NO: 64. In another embodiment, the antibody or antigen bindingfragment thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30amino acid substitutions in the variable heavy light chain of SEQ IDNO:63 and/or the variable light chain of SEQ ID NO: 64. In anotherembodiment, the antibody or antigen binding fragment thereof comprises1, 2 or 3 amino acid substitutions in the heavy chain CDRs (SEQ ID NOs:57-59) or in the light chain CDRs (SEQ ID NOs: 60-62).

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that binds to the same epitope of human TIGITas an antibody comprising the variable heavy chain of SEQ ID NO: 94 andthe variable light chain of SEQ ID NO:95, wherein the antibody orantigen binding fragment thereof has at least one of the followingcharacteristics: (i) binds to human TIGIT with a KD value of about1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmon resonance(e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); (ii)cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocks binding ofhuman TIGIT to human CD155 and human CD112; (iv) increases T cellactivation; (v) stimulates antigen-specific T-cell production of IL-2and IFNγ; (vi) blocks induction of T cell suppression of activationinduced by TIGIT ligation with cognate ligands CD155 and CD112. In oneembodiment, the antibody or antigen binding fragment thereof comprisesat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence with thevariable heavy chain of any one of SEQ ID NOs:94 or 124-129, and/or thevariable light chain of any one of SEQ ID NO: 95 or 130-133. In anotherembodiment, the antibody or antigen binding fragment thereof comprises1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions in thevariable heavy light chain of any one of SEQ ID NOs:94 or 124-129,and/or the variable light chain of any one of SEQ ID NO: 95 or 130-133.In another embodiment, the antibody or antigen binding fragment thereofcomprises 1, 2 or 3 amino acid substitutions in the heavy chain CDRs(SEQ ID NOs: 88-90) and/or in the light chain CDRs (SEQ ID NOs: 91-93).In another embodiment, the antibody or antigen binding fragment thereofcomprises heavy chain CDRs of SEQ ID NOs: 88, 134 and 90 and/or thelight chain CDRs of SEQ ID NOs: 91, 92 and 93.

In one embodiment, the invention provides an antibody or antigen bindingfragment thereof that binds to an epitope of human TIGIT that comprisesat least one of the following regions: residues 53-57, residues 60-65,residues 68-70, residues 72-81, residues 94-95, and residues 109-119 ofSEQ ID NO:31. In one embodiment, the invention provides an antibody orantigen binding fragment thereof that binds to an epitope of human TIGITcomprising residues: 53-57, 60-65, 68-70, 72-81, 94-95, and 109-119 ofSEQ ID NO:31.

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that cross-blocks the binding of (or competeswith) an antibody comprising the variable heavy chain of SEQ ID NO: 7and the variable light chain of SEQ ID NO: 8 to human TIGIT, wherein theantibody or antigen binding fragment thereof has at least one of thefollowing characteristics: (i) binds to human TIGIT with a KD value ofabout 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET);(ii) cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocksbinding of human TIGIT to human CD155 and human CD112; (iv) increases Tcell activation; (v) stimulates antigen-specific T-cell production ofIL-2 and IFNγ; (vi) blocks induction of T cell suppression of activationinduced by TIGIT ligation with cognate ligands CD155 and CD112. In oneembodiment, the antibody or antigen binding fragment thereof comprisesat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity withthe variable heavy chain or variable light chains of SEQ ID NOs: 7-30 or37-52. In another embodiment, the antibody or antigen binding fragmentthereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acidsubstitutions in the variable heavy light chains of SEQ ID NOs:7, 9-24and 37-47 or the variable light chains of SEQ ID NOs: 8, 25-30 and48-52. In another embodiment, the antibody or antigen binding fragmentthereof comprises 1, 2 or 3 amino acid substitutions in the heavy chainCDRs (SEQ ID NOs: 1-3) or in the light chain CDRs (SEQ ID NOs: 3-6).

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that cross-blocks the binding of (or competeswith) an antibody comprising the variable heavy chain of SEQ ID NO: 63and the variable light chain of SEQ ID NO: 64 to human TIGIT, whereinthe antibody or antigen binding fragment thereof has at least one of thefollowing functional characteristics: (i) binds to human TIGIT with a KDvalue of about 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surfaceplasmon resonance (e.g., BIACORE) or a similar technique (e g KinExa orOCTET); (ii) cross-reacts with cynomolgous and rhesus TIGIT; (iii)blocks binding of human TIGIT to human CD155 and human CD112; (iv)increases T cell activation; (v) stimulates antigen-specific T-cellproduction of IL-2 and IFNγ (vi) blocks induction of T cell suppressionof activation induced by TIGIT ligation with cognate ligands CD155 andCD112. In one embodiment, the antibody comprises at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity with the variable heavychain of SEQ ID NO:63 or the variable light chains of SEQ ID NO: 64. Inanother embodiment, the antibody or antigen binding fragment thereofcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acidsubstitutions in the variable heavy light chain of SEQ ID NO: 63 or thevariable light chain of SEQ ID NO: 64. In another embodiment, theantibody or antigen binding fragment thereof comprises 1, 2 or 3 aminoacid substitutions in the heavy chain CDRs (SEQ ID NOs: 57-59) or in thelight chain CDRs (SEQ ID NOs: 60-62).

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that cross-blocks the binding of (or competeswith) an antibody comprising the variable heavy chain of SEQ ID NO: 94and the variable light chain of SEQ ID NO: 95 to human TIGIT, whereinthe antibody or antigen binding fragment thereof has at least one of thefollowing characteristics: (i) binds to human TIGIT with a KD value ofabout 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET);(ii) cross-reacts with cynomolgous and rhesus TIGIT; (iii) blocksbinding of human TIGIT to human CD155 and human CD112; (iv) increases Tcell activation; (v) stimulates antigen-specific T-cell production ofIL-2 and IFNγ (vi) blocks induction of T cell suppression of activationinduced by TIGIT ligation with cognate ligands CD155 and CD112. In oneembodiment, the antibody comprises at least 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% sequence identity with the variable heavy chain of SEQID NO: 94 or the variable light chains of SEQ ID NO: 95. In anotherembodiment, the antibody or antigen binding fragment thereof comprises1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid substitutions in thevariable heavy light chain of any one of SEQ ID NOs: 94 or 124-129, orthe variable light chain of any one of SEQ ID NOs: 95 or 130-133. Inanother embodiment, the antibody or antigen binding fragment thereofcomprises 1, 2 or 3 amino acid substitutions in the heavy chain CDRs(SEQ ID NOs: 88-90) or in the light chain CDRs (SEQ ID NOs: 91-93). Inone embodiment, the antibody or antigen binding fragment thereofcomprises: a heavy chain variable region comprising SEQ ID NO:128 and alight chain variable region comprising SEQ ID NO:132. In anotherembodiment, antibody or antigen binding fragment thereof comprises: aheavy chain variable region comprising SEQ ID NO:127 and a light chainvariable region comprising SEQ ID NO:130. In another embodiment,antibody or antigen binding fragment thereof comprises: a heavy chainvariable region comprising SEQ ID NO:128 and a light chain variableregion comprising SEQ ID NO:133.

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that binds to human TIGIT comprising a variableheavy chain selected from the group consisting of any one of SEQ ID NOs:9-24 and 37-47, and/or a variable light chain selected from the groupconsisting of any one of SEQ ID NOs: 25-30 and 48-52. In one embodiment,the antibody comprises an amino acid substitution in FR4 of the heavychain, wherein the substitution is made at position 122 of SEQ ID NOs:9-24 and 37-47, wherein the residue is substituted from M to: V, L, A,R, N, P, Q, E, G, I, H, K, F, S, T, W or Y. In one embodiment, theantibody comprises two amino acid substitutions in FR4 of the heavychain, wherein the substitutions are made at positions 122 and 123 ofSEQ ID NOs: 9-24 and 37-47, wherein the residues are substituted from Mand V to T and L, respectively.

In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that binds to human TIGIT comprising a variableheavy chain selected from the group consisting of any one of SEQ ID NOs:9-24 and 37-47, and/or a variable light chain selected from the groupconsisting of any one of SEQ ID NOs: 25-30 and 48-52. In one embodiment,the antibody comprises an amino acid substitution in FR4 of the heavychain, wherein the substitution is made at position 122 of SEQ ID NOs:9-24 and 37-47, wherein the residue is substituted from M to: V, L, A,R, N, P, Q, E, G, I, H, K, F, S, T, W or Y. In one embodiment, theantibody comprises two amino acid substitutions in FR4 of the heavychain, wherein the substitutions are made at positions 122 and 123 ofSEQ ID NOs: 9-24 and 37-47, wherein the residues are substituted from Mand V to T and L, respectively.

In one embodiment, the invention relates to an isolated antibody orantigen binding fragment that binds to human TIGIT comprising: a heavychain comprising the amino acid sequence of SEQ ID NO: 7 or variantthereof comprising up to 30 amino acid substitutions, and/or a lightchain comprising the amino acid sequence of SEQ ID NO: 8 comprising upto 12 amino acid substitutions. In one embodiment, the heavy chaincomprises the amino acid sequence of SEQ ID NO: 7 comprising amino acidsubstitutions at one or more positions selected from the groupconsisting of: 6, 9, 12, 15, 16, 17, 23, 25, 37, 39, 40, 43, 44, 45, 48,67, 68, 70, 71, 79, 81, 83, 87, 88, 92, 94, 119, 122 and 123. In oneembodiment, the heavy chain comprises the amino acid sequence of SEQ IDNO:7 comprising amino acid substitutions at one or more positionsselected from the group consisting of: 6, 9, 12, 15, 16, 17, 23, 25, 37,39, 40, 43, 44, 45, 48, 67, 68, 70, 71, 79, 81, 83, 87, 88, 92, 94, 110,119, 122 and 123, wherein: the amino acid at position 6 can be E or Q,the amino acid at position 9 can be P or A, the amino acid at position12 can be V or L, the amino acid at position 15 can be S or P, the aminoacid at position 16 can be Q or E or G, the amino acid at position 17can be S or T, the amino acid at position 23 can be S or T, the aminoacid at position 25 can be T or S, the amino acid at position 37 can beI or V, the amino acid at position 39 can be K or Q, the amino acid atposition 40 can be F or P, the amino acid at position 43 can be N or K,the amino acid at position 44 can be K or G, the amino acid at position45 can be M or L, the amino acid at position 48 can be M or I, the aminoacid at position 67 can be I or V, the amino acid at position 68 can beS or T, the amino acid at position 70 can be T or S, the amino acid atposition 71 can be R or V, the amino acid at position 79 can be F or S,the amino acid at position 81 can be Q or K, the amino acid at position83 can be H or S, the amino acid at position 87 can be T or A, the aminoacid at position 88 can be D or A, the amino acid at position 92 can beT or V, the amino acid at position 94 can be S or Y, the amino acid atposition 110 can be W, F, Y, I, V or L, the amino acid at position 119can be P or Q, the amino acid at position 122 can be M, V, L, A, R, N,P, Q, E, G, I, H, K, F, S, T, W or Y, and the amino acid at position 123can be V, T or L. In one embodiment, the light chain comprises the aminoacid sequence of SEQ ID NO: 8 comprising amino acid substitutions at oneor more positions selected from the group consisting of: 10, 21, 22, 40,42, 46, 52, 53, 58, 77, 83, 87, 95 and 106. In one embodiment, the lightchain comprises the amino acid sequence of SEQ ID NO: 8 comprising aminoacid substitutions at one or more positions selected from the groupconsisting of: 10, 21, 22, 40, 42, 46, 52, 53, 58, 77, 83, 87, 95 and106 wherein: the amino acid at position 10 can be L or S, the amino acidat position 21 can be L or I, the amino acid at position 22 can be N orT, the amino acid at position 40 can be L or P, the amino acid atposition 42 can be E or K, the amino acid at position 46 can be F or L,the amino acid at position 52 can be N, S, T, G or D, the amino acid atposition 53 can be S, N, T, Y or Q, the amino acid at position 58 can beI or V, the amino acid at position 77 can be G or S, the amino acid atposition 83 can be V or F, the amino acid at position 87 can be F or Y,the amino acid at position 95 can be W, F, Y, I, V or L, and the aminoacid at position 105 can be L or I.

In another embodiment, the invention relates to an antibody or antigenbinding fragment that binds to human TIGIT comprising: a heavy chaincomprising the amino acid sequence of SEQ ID NO: 9 and/or a light chaincomprising the amino acid sequence of SEQ ID NO: 25, wherein each of thevariable chains can comprise 1, 2, 3, 4, 5, 6, 7 or 8 amino acidsubstitutions. In one embodiment, the antibody can comprise mutations atpositions 27, 48, 67, 71, 83, 110, 122 and 123 of SEQ ID NO: 9. Forexample, with respect to SEQ ID NO: 9: the amino acid at position 27 canbe G or S; the amino acid at position 48 can be I or M; the amino acidat position 67 can be V or I; the amino acid at position 71 can be V orR; the amino acid at position 83 can be S or H; the amino acid atposition 110 can be W, F, Y, I, V or L; the amino acid at position 122can be M, V, L, A, R, N, P, Q, E, G, I, H, K, F, S, T, W or Y; and theamino acid at position 123 can be V, T or L. In another embodiment, theantibody can comprise mutations at positions 46, 52, 53, 58 and 95 ofSEQ ID NO: 25. For example, with respect to SEQ ID NO: 25, the aminoacid at position 46 can be L or F; the amino acid at position 52 can beN, S, T, G or D, the amino acid at position 53 can be S, N, T, Y or Q;the amino acid at position 58 can be I or V; the amino acid at position58 can be V or I; and the amino acid at position 95 can be W, F, Y, I, Vor L.

In one embodiment, the invention relates to an antibody or antigenbinding fragment that binds to human TIGIT comprising: a heavy chaincomprising the amino acid sequence of SEQ ID NO: 94 or variant thereofcomprising up to 30 amino acid substitutions, and a light chaincomprising the amino acid sequence of SEQ ID NO: 95 or a variant thereofcomprising up to 18 amino acid substitutions. In one embodiment, theheavy chain comprises a variant of the amino acid sequence of SEQ ID NO:94 comprising amino acid substitutions at one or more positions selectedfrom the group consisting of: 5, 9, 11, 12, 16, 20, 38, 40, 44, 56, 57,61, 62, 65, 67, 68, 72, 74, 76, 79, 85, 87, 89, 91, 92, 104 and 111. Inone embodiment, the heavy chain comprises a variant of the amino acidsequence of SEQ ID NO: 94 comprising amino acid substitutions at one ormore positions selected from the group consisting of: 5, 9, 11, 12, 16,20, 38, 40, 44, 56, 57, 61, 62, 65, 67, 68, 72, 74, 76, 79, 85, 87, 89,91, 92, 104 and 111, wherein: the amino acid at position 5 can be Q orV, the amino acid at position 9 can be P or A, the amino acid atposition 11 can be V or L, the amino acid at position 12 can be V or K,the amino acid at position 16 can be S or A, the amino acid at position20 can be M or V, the amino acid at position 38 can be K or R, the aminoacid at position 40 can be K or A, the amino acid at position 44 can beG or R, the amino acid at position 56 can be D, R, L, K, F, S, Y or V,the amino acid at position 57 can be G, R, N, Q, E, L K, S, Y or V, theamino acid at position 61 can be N, A or S, the amino acid at position62 can be E or Q, the amino acid at position 65 can be K or Q, the aminoacid at position 67 can be R or K, the amino acid at position 68 can beA or V, the amino acid at position 72 can be S or R, the amino acid atposition 74 can be K or T, the amino acid at position 76 can be S, I, Aor T, the amino acid at position 79 can be A or V, the amino acid atposition 85 can be R or S, the amino acid at position 87 can be T or R,the amino acid at position 89 can be D or E, the amino acid at position91 can be S or T, the amino acid at position 92 can be A or V, the aminoacid at position 104 can be W, A, D, E, F, G, I, K, N, Q, R, S, T, V orY, and the amino acid at position 111 can be A or Q. In one embodiment,the light chain comprises a variant of the amino acid sequence of SEQ IDNO: 95 comprising amino acid substitutions at one or more positionsselected from the group consisting of: 9, 17, 18, 40, 43, 45, 48, 50,51, 70, 72, 74, 76, 83, 84, 100, 103 and 106. In one embodiment, thelight chain comprises a variant of the amino acid sequence of SEQ ID NO:95 comprising amino acid substitutions at one or more positions selectedfrom the group consisting of: 9, 17, 18, 40, 43, 45, 48, 50, 51, 70, 72,74, 76, 83, 84, 100, 103 and 106, wherein the amino acid at position 9can be A or S, the amino acid at position 17 can be E or D, the aminoacid at position 18 can be T or R, the amino acid at position 40 can beQ or P, the amino acid at position 43 can be S, A or V, the amino acidat position 45 can be Q or K, the amino acid at position 48 can be V orI, the amino acid at position 50 can be N, A, Y, W, S, T, I or V, theamino acid at position 51 can be A, N, I, L, T or V, the amino acid atposition 70 can be Q or D, the amino acid at position 72 can be S or T,the amino acid at position 74 can be K or T, the amino acid at position76 can be N or S, the amino acid at position 83 can be F or V, the aminoacid at position 84 can be G or A, the amino acid at position 100 can beA or Q, the amino acid at position 103 can be T or R and the amino acidat position at position 106 can be L or I.

In one embodiment, the invention relates to an antibody or antigenbinding fragment that binds to human TIGIT comprising: a heavy chaincomprising the amino acid sequence of SEQ ID NO: 124 or variant thereofcomprising up to 10 amino acid substitutions, and/or a light chaincomprising the amino acid sequence of SEQ ID NO: 130 or a variantthereof comprising up to 5 amino acid substitutions. In anotherembodiment, the invention provides an antibody or antigen bindingfragment thereof that binds to human TIGIT comprising a variable heavychain of SEQ ID NO: 124 or a variant thereof, wherein the variantcomprises substitutions at one or more positions selected from the groupconsisting of: 16, 44, 56, 57, 61, 72, 74, 76, 79, 85, 89, 92 and 104.In another embodiment, the invention provides an antibody or antigenbinding fragment thereof that binds to human TIGIT comprising a variableheavy chain of SEQ ID NO: 124 or a variant thereof, wherein the variantcomprises substitutions at one or more positions selected from the groupconsisting of: 16, 44, 56, 57, 61, 72, 74, 76, 79, 85, 89, 92 and 104,wherein the amino acid at position 16 can be A or S, the amino acid atposition 44 can be R or G, the amino acid at position 56 can be D, R, L,K, F, S, Y or V, the amino acid at position 57 can be G, R, N, Q, E, LK, S, Y or V, the amino acid at position 61 can be S or A, the aminoacid at position 72 can be R or S, the amino acid at position 74 can beT or K, the amino acid at position 76 can be A or T or I, the amino acidat position 79 can be A or V, the amino acid at position 85 can be S orR, the amino acid at position 89 can be E or D, the amino acid atposition 92 can be A or V and the amino acid at position 104 can be W,A, D, E, F, G, I, K, N, Q, R, S, T, V or Y. In another embodiment, theinvention provides an antibody or antigen binding fragment thereof thatbinds to human TIGIT comprising a variable light chain of SEQ ID NO: 130or a variant thereof comprising substitutions at one or more positionsselected from the group consisting of: 43, 50, 51, 70 and 83. In anotherembodiment, the invention provides an antibody or antigen bindingfragment thereof that binds to human TIGIT comprising a variable lightchain of SEQ ID NO: 130 or a variant thereof comprising substitutions atone or more positions selected from the group consisting of: 43, 50, 51,70 and 83, wherein the amino acid at position 43 can be S, A or V, theamino acid at position 50 can be N, A, Y, W, S, T, I or V, the aminoacid at position 51 can be A, N, I, L, T or V, the amino acid atposition 70 can be Q or D, and the amino acid at position 83 can be F orV.

In any of the above embodiments, the antibody or antigen bindingfragment thereof is isolated.

In any of the above embodiments, the antibody or antigen bindingfragment thereof is a recombinant antibody.

In any of the above embodiments, the antibody or antigen bindingfragment thereof is a full-length antibody.

In any of the above embodiments, the antibody or antigen bindingfragment thereof is a humanized antibody.

In any of the above embodiments, the antibody or antigen bindingfragment thereof is a humanized antibody comprising two heavy chains andtwo light chains. In one embodiment, the heavy chains are of the IgG1isotype and the light chains are kappa light chains.

In any of the above mentioned embodiments, the antibody or antigenbinding fragment thereof of the invention can comprise a variable heavyregion consisting of: (a) any of the variable heavy chains describedabove and (b) a leader peptide (for example, the leader peptide of SEQID NO: 53). In any of the above mentioned embodiments, the antibody orantigen binding fragment thereof of the invention can comprise a lightheavy region consisting of: (a) any of the light heavy chains describedabove and (b) a leader peptide (for example, the leader peptide of SEQID NO: 54).

In any of the above mentioned embodiments, the antibody or antigenbinding fragment thereof of the invention is an antibody comprising anyof the variable heavy chains described above and any human heavy chainconstant domain. In one embodiment, the antibody or antigen bindingfragment thereof of the invention is of the IgG isotype, and comprises ahuman IgG1, IgG2, IgG3 or IgG4 human heavy chain constant domain. In oneembodiment, the antibody or antigen binding fragment thereof of theinvention comprises a human heavy chain IgG1 constant domain (SEQ ID NO:86) or a variant thereof, wherein the variant comprises up to 20modified amino acid substitutions. In one embodiment, the antibody orantigen binding fragment thereof of the invention is an antibodycomprising a human heavy chain IgG1 constant domain comprising the aminoacid sequence of SEQ ID NO: 86. In one embodiment, the antibody orantigen binding fragment thereof of the invention comprises a humanheavy chain IgG1 constant domain wherein the IgG1 constant domain isafucosylated. In one embodiment, the antibody or antigen bindingfragment thereof of the invention comprises a human heavy chain IgG4constant domain or a variant thereof, wherein the variant comprises upto 20 modified amino acid substitutions. In another embodiment, theantibody or antigen binding fragment thereof of the invention comprisesa human heavy chain IgG4 constant domain, wherein the amino acid atposition 228 (using EU numbering scheme) has been substituted from Serto Pro. In one embodiment, the antibody or antigen binding fragmentthereof of the invention comprises a human heavy chain IgG4 constantdomain comprising the amino acid sequence of SEQ ID NO: 55.

In any of the above mentioned embodiments, the antibody or antigenbinding fragment thereof of the invention can comprise any of thevariable light chains described above and human light chain constantdomain. In one embodiment, the antibody or antigen binding fragmentthereof of the invention comprises a human kappa light chain constantdomain or a variant thereof, wherein the variant comprises up to 20modified amino acid substitutions. In another embodiment, the antibodyor antigen binding fragment thereof of the invention comprises a humanlambda light chain constant domain or a variant thereof, wherein thevariant comprises up to 20 modified amino acid substitutions. In oneembodiment, the antibody or antigen binding fragment thereof of theinvention comprises a human kappa light chain constant domain comprisingthe amino acid sequence of SEQ ID NO: 56.

In one embodiment, the anti-TIGIT antibody of the invention comprises afull tetrameric structure having two light chains and two heavy chains,wherein each light chain comprises: a variable region comprising SEQ IDNO:132 and a human kappa light chain (SEQ ID NO:56); and each heavychain comprises: a variable region comprising SEQ ID NO:128, a humanIgG1 constant region (SEQ ID NO:86).

In one embodiment, the anti-TIGIT antibody of the invention comprises afull tetrameric structure having two light chains and two heavy chains,wherein each light chain comprises: a variable region comprising SEQ IDNO:130 and a human kappa light chain (SEQ ID NO:56); and each heavychain comprises: a variable region comprising SEQ ID NO:127, a humanIgG1 constant region (SEQ ID NO:86).

In one embodiment, the anti-TIGIT antibody of the invention comprises afull tetrameric structure having two light chains and two heavy chains,wherein each light chain comprises: a variable region comprising SEQ IDNO:133 and a human kappa light chain (SEQ ID NO:56); and each heavychain comprises: a variable region comprising SEQ ID NO:128, a humanIgG1 constant region (SEQ ID NO:86).

In any of the above mentioned embodiments, the anti-TIGIT antibody orantigen binding fragment thereof of the invention can be conjugated toat least one therapeutic agent. In one embodiment, the wherein thetherapeutic agent comprises a second antibody or fragment thereof, animmunomodulator, a hormone, a cytotoxic agent, an enzyme, aradionuclide, a second antibody conjugated to at least oneimmunomodulator, enzyme, radioactive label, hormone, antisenseoligonucleotide, or cytotoxic agent, or a combination thereof.

The invention also provides isolated polypeptides comprising the aminoacid sequence of any one of SEQ ID NOs: 1-30, 37-52, 57-83 or 88-151, ora fragment of any said sequences.

The invention also provides isolated nucleic acids encoding anyone ofthe anti-TIGIT antibodies or antigen binding fragments of the invention.In one embodiment, the invention provides isolated nucleic acidsencoding anyone of the polypeptides of SEQ ID NOs: 1-30, 37-52, 57-64 or88-133, wherein said polypeptides can optionally comprise a leadersequence. The invention also provides expression vectors comprising anucleic acid encoding anyone of the polypeptides of SEQ ID NOs: 1-30,37-52, 57-83 or 88-167 (wherein said polypeptides can optionallycomprise a leader sequence). These isolated nucleic acids and theexpression vectors comprising them may be used to express the antibodiesof the invention or antigen binding fragments thereof in recombinanthost cells. Thus, the invention also provides host cells comprisingisolated nucleic acids encoding anyone of the polypeptides of SEQ IDNOs: 1-30, 37-52, 57-83 or 88-151 (wherein said polypeptides canoptionally comprise a leader sequence). In one embodiment, the host cellis Chinese hamster ovary cell. In one embodiment, the host cell is ayeast cell, for example a Pichia cell or a Pichia pastoris host cell.

The invention also provides pharmaceutical compositions comprising anantibody or antigen binding fragment of the invention and apharmaceutically acceptable carrier or diluent. In one embodiment, thecomposition comprises a further therapeutic agent. In one embodiment,the further therapeutic agent is selected from the group consisting of:an anti-PD1 antibody or an antigen binding fragment thereof; ananti-LAG3 antibody or an antigen binding fragment thereof; an anti-VISTAantibody or an antigen binding fragment thereof; an anti-BTLA antibodyor an antigen binding fragment thereof; an anti-TIM3 antibody or anantigen binding fragment thereof; an anti-CTLA4 antibody or an antigenbinding fragment thereof; an anti-HVEM antibody or an antigen bindingfragment thereof; an anti-CD27 antibody or an antigen binding fragmentthereof; an anti-CD137 antibody or an antigen binding fragment thereof;an anti-OX40 antibody or an antigen binding fragment thereof; ananti-CD28 antibody or an antigen binding fragment thereof; an anti-PDL1antibody or an antigen binding fragment thereof; an anti-PDL2 antibodyor an antigen binding fragment thereof; an anti-GITR antibody or anantigen binding fragment thereof; an anti-ICOS antibody or an antigenbinding fragment thereof; an anti-SIRPα antibody or an antigen bindingfragment thereof; an anti-ILT2 antibody or antigen binding fragmentthereof; an anti-ILT3 antibody or antigen binding fragment thereof; ananti-ILT4 antibody or antigen binding fragment thereof; and an anti-ILT5antibody or an antigen binding fragment thereof; an anti 4-1BB antibodyor an antigen binding fragment thereof. In one embodiment, the anti-PD1antibody or an antigen binding fragment thereof; is selected from thegroup consisting of: pembrolizumab or an antigen binding fragmentthereof and nivolumab or an antigen binding fragment thereof. In oneembodiment, the anti-TIGIT antibody or antigen binding fragment of theinvention comprises: (i) a heavy chain variable region CDR1 comprisingthe amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variableregion CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) aheavy chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 3, 79, 80, 81, 82, 83 or 140; (iv) a light chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) alight chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 5, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 141; and (vi) alight chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 6, 74, 75, 76, 77, 78 or 142. In one embodiment, theanti-TIGIT antibody or antigen binding fragment thereof comprises: (i) aheavy chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 88; (ii) a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 89, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 134, 135 or 147; (iii) aheavy chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 90; (iv) a light chain variable region CDR1 comprising theamino acid sequence of SEQ ID NO: 91; (v) a light chain variable regionCDR2 comprising the amino acid sequence of SEQ ID NO: 92, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122 or 148; and (vi) a light chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO:93. In one embodiment, the anti-TIGIT antibody or antigen bindingfragment of the invention comprises: (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavychain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 2; (iii) a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5;and (vi) a light chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 6. In another embodiment, the anti-TIGIT antibodyor antigen binding fragment of the invention comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 57; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 58; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 59; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:60; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 61; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 62. In one embodiment,the anti-TIGIT antibody or antigen binding fragment thereof comprises:(i) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 89, 134 or 135; (iii) aheavy chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 90; (iv) a light chain variable region CDR1 comprising theamino acid sequence of SEQ ID NO: 91; (v) a light chain variable regionCDR2 comprising the amino acid sequence of SEQ ID NO: 92; and (vi) alight chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 93. In one embodiment, the anti-TIGIT antibody or antigenbinding fragment thereof comprises: (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavychain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 134; (iii) a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 90; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 91; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:92; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 93.

In one embodiment, the invention provides a composition comprising: (i)an anti-TIGIT antibody or antigen binding fragment of the invention; and(ii) an anti-PD1 antibody comprising the heavy chain sequence of SEQ IDNO: 33 and the light chain variable sequence of SEQ ID NO: 34. Inanother embodiment, the invention provides a composition comprising: (a)an anti-TIGIT antibody or antigen binding fragment of the invention; and(b) an anti-PD1 antibody comprising the heavy chain sequence of SEQ IDNO: 35 and the light chain variable sequence of SEQ ID NO: 36. In oneembodiment, the anti-PD1 antibody is administered prior to theadministration of an anti-TIGIT antibody. In one embodiment, theanti-PD1 antibody is administered 4-10 days prior to the administrationof the anti-TIGIT antibody. In one embodiment, pretreatment treatmentwith anti-PD1 antibody may modulate immune cells resulting in enhancedFc-mediated function of the anti-TIGIT antibodies. In one embodiment,the anti-TIGIT antibody or antigen binding fragment of the inventioncomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3,79, 80, 81, 82, 83 or 140; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5,65, 66, 67, 68, 69, 70, 71, 72, 73 or 141; and (vi) a light chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6,74, 75, 76, 77, 78 or 142. In one embodiment, the anti-TIGIT antibody orantigen binding fragment thereof comprises: (i) a heavy chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 88; (ii) aheavy chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 89, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 134, 135 or 147; (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 91; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 92, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122 or 148; and (vi) a light chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 93. In one embodiment, theanti-TIGIT antibody or antigen binding fragment of the inventioncomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3;(iv) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 6. In another embodiment, the anti-TIGIT antibody or antigen bindingfragment of the invention comprises: (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 57; (ii) a heavychain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 58; (iii) a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 59; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 60; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:61; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 62. In one embodiment, the anti-TIGITantibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 89, 134 or 135; (iii) a heavy chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) alight chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 91; (v) a light chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 92; and (vi) a light chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In oneembodiment, the anti-TIGIT antibody or antigen binding fragment thereofcomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 90; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 92; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 93.

The invention also comprises a combination comprising an anti-TIGITantibody or antigen binding fragment of the invention, in combinationwith one, two or more therapeutic agents; wherein the second therapeuticagent is selected from the group consisting of: an anti-PD1 antibody oran antigen binding fragment thereof; an anti-LAG3 antibody or an antigenbinding fragment thereof; an anti-VISTA antibody or an antigen bindingfragment thereof; an anti-BTLA antibody or an antigen binding fragmentthereof; an anti-TIM3 antibody or an antigen binding fragment thereof;an anti-CTLA4 antibody or an antigen binding fragment thereof; ananti-HVEM antibody or an antigen binding fragment thereof; an anti-CD27antibody or an antigen binding fragment thereof; an anti-CD137 antibodyor an antigen binding fragment thereof; an anti-OX40 antibody or anantigen binding fragment thereof; an anti-CD28 antibody or an antigenbinding fragment thereof; an anti-PDL1 antibody or an antigen bindingfragment thereof; an anti-PDL2 antibody or an antigen binding fragmentthereof; an anti-GITR antibody or an antigen binding fragment thereof;an anti-ICOS antibody or an antigen binding fragment thereof; ananti-SIRPα antibody or an antigen binding fragment thereof; an anti-ILT2antibody or antigen binding fragment thereof; an anti-ILT3 antibody orantigen binding fragment thereof; an anti-ILT4 antibody or antigenbinding fragment thereof; an anti-ILT5 antibody or an antigen bindingfragment thereof; and an anti 4-1BB antibody or an antigen bindingfragment thereof. In one embodiment, the anti-TIGIT antibody or antigenbinding fragment of the invention comprises: (i) a heavy chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) aheavy chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising theamino acid sequence of SEQ ID NO: 3, 79, 80, 81, 82, 83 or 140; (iv) alight chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 4; (v) a light chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 5, 65, 66, 67, 68, 69, 70, 71, 72, 73or 141; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 6, 74, 75, 76, 77, 78 or 142. In oneembodiment, the anti-TIGIT antibody or antigen binding fragment thereofcomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 89, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 134, 135 or147; (iii) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 90; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 91; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:92, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 or 148; and(vi) a light chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 93. In one embodiment, the anti-TIGIT antibody orantigen binding fragment of the invention comprises: (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1;(ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4;(v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 6. In anotherembodiment, the anti-TIGIT antibody or antigen binding fragment of theinvention comprises: (i) a heavy chain variable region CDR1 comprisingthe amino acid sequence of SEQ ID NO: 57; (ii) a heavy chain variableregion CDR2 comprising the amino acid sequence of SEQ ID NO: 58; (iii) aheavy chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 59; (iv) a light chain variable region CDR1 comprising theamino acid sequence of SEQ ID NO: 60; (v) a light chain variable regionCDR2 comprising the amino acid sequence of SEQ ID NO: 61; and (vi) alight chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 62. In one embodiment, the anti-TIGIT antibody or antigenbinding fragment thereof comprises: (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavychain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 89, 134 or 135; (iii) a heavy chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 90; (iv) a light chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 91; (v) alight chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 92; and (vi) a light chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 93. In one embodiment, theanti-TIGIT antibody or antigen binding fragment thereof comprises: (i) aheavy chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 88; (ii) a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 134; (iii) a heavy chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) alight chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 91; (v) a light chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 92; and (vi) a light chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 93.

The invention also provides a vessel or injection device comprisinganyone of the anti-TIGIT antibodies or antigen binding fragments of theinvention. In one embodiment, the anti-TIGIT antibody or antigen bindingfragment of the invention comprises: (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavychain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 2; (iii) a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, 79, 80, 81, 82, 83 or 140; (iv) a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 4; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 5, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 141; and(vi) a light chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 6, 74, 75, 76, 77, 78 or 142. In one embodiment,the anti-TIGIT antibody or antigen binding fragment thereof comprises:(i) a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 89, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 134, 135 or147; (iii) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 90; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 91; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:92, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 or 148; and(vi) a light chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 93. In one embodiment, the anti-TIGIT antibody orantigen binding fragment of the invention comprises: (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1;(ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4;(v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 6. In anotherembodiment, the anti-TIGIT antibody or antigen binding fragment of theinvention comprises: (i) a heavy chain variable region CDR1 comprisingthe amino acid sequence of SEQ ID NO: 57; (ii) a heavy chain variableregion CDR2 comprising the amino acid sequence of SEQ ID NO: 58; (iii) aheavy chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 59; (iv) a light chain variable region CDR1 comprising theamino acid sequence of SEQ ID NO: 60; (v) a light chain variable regionCDR2 comprising the amino acid sequence of SEQ ID NO: 61; and (vi) alight chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 62. In one embodiment, the anti-TIGIT antibody or antigenbinding fragment thereof comprises: (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavychain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 89, 134 or 135; (iii) a heavy chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 90; (iv) a light chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 91; (v) alight chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 92; and (vi) a light chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 93. In one embodiment, theanti-TIGIT antibody or antigen binding fragment thereof comprises: (i) aheavy chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 88; (ii) a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 134; (iii) a heavy chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) alight chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 91; (v) a light chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 92; and (vi) a light chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 93.

The invention also provides a method of producing an anti-TIGIT antibodyor antigen binding fragment of the invention comprising: culturing ahost cell comprising a polynucleotide encoding a heavy chain and/orlight chain of an antibody of the invention (or an antigen bindingfragment thereof) under conditions favorable to expression of thepolynucleotide; and optionally, recovering the antibody or antigenbinding fragment from the host cell and/or culture medium. In oneembodiment, the polynucleotide encoding the heavy chain and thepolynucleotide encoding the light chain are in a single vector. Inanother embodiment, the polynucleotide encoding the heavy chain and thepolynucleotide encoding the light chain are in different vectors. In oneembodiment, the polynucleotide encoding the heavy chain and thepolynucleotide encoding the light chain encode an antibody or antigenbinding fragment comprising: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2;(iii) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 3, 79, 80, 81, 82, 83 or 140; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:4;(v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO:5, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 141; and(vi) a light chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO:6, 74, 75, 76, 77, 78 or 142. In anotherembodiment, the polynucleotide encoding the heavy chain and thepolynucleotide encoding the light chain encode an antibody or antigenbinding fragment comprising: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO:88; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:89, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 134, 135 or 147; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 90; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:91; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 92, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122 or 148; and (vi) a light chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 93. In another embodiment, thepolynucleotide encoding the heavy chain and the polynucleotide encodingthe light chain encode an antibody or antigen binding fragmentcomprising: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3;(iv) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 6. In another embodiment, the polynucleotide encoding the heavychain and the polynucleotide encoding the light chain encode an antibodyor antigen binding fragment comprising: (i) a heavy chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 57; (ii) aheavy chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 58; (iii) a heavy chain variable region CDR3 comprising theamino acid sequence of SEQ ID NO: 59; (iv) a light chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 60; (v) a lightchain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 61; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO:62. In another embodiment, the polynucleotideencoding the heavy chain and the polynucleotide encoding the light chainencode an antibody or antigen binding fragment comprising: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 89, 134 or 135; (iii) a heavy chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) alight chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 91; (v) a light chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 92; and (vi) a light chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 93. Inanother embodiment, the polynucleotide encoding the heavy chain and thepolynucleotide encoding the light chain encode an antibody or antigenbinding fragment comprising: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:134; (iii) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 90; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 91; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:92; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 93. In another embodiment, thepolynucleotide encoding the heavy chain and the polynucleotide encodingthe light chain encode an antibody or antigen binding fragmentcomprising: a heavy chain variable region comprising SEQ ID NO:128 and alight chain variable region comprising SEQ ID NO:132. In anotherembodiment, the polynucleotide encoding the heavy chain and thepolynucleotide encoding the light chain encode an antibody or antigenbinding fragment comprising: a heavy chain variable region comprisingSEQ ID NO:127 and a light chain variable region comprising SEQ IDNO:130. In another embodiment, the polynucleotide encoding the heavychain and the polynucleotide encoding the light chain encode an antibodyor antigen binding fragment comprising: a heavy chain variable regioncomprising SEQ ID NO:128 and a light chain variable region comprisingSEQ ID NO:133.

The invention also provides a method of treating cancer in a subject inneed thereof, comprising administering to the subject an effectiveamount of an anti-TIGIT antibody or antigen binding fragment of theinvention, optionally in association with a further therapeutic agent ortherapeutic procedure. In one embodiment, the subject been treated is ahuman subject. In one embodiment, the further therapeutic agent isselected from the group consisting of: an anti-PD1 antibody or anantigen binding fragment thereof; an anti-LAG3 antibody or an antigenbinding fragment thereof; an anti-VISTA antibody or an antigen bindingfragment thereof; an anti-BTLA antibody or an antigen binding fragmentthereof; an anti-TIM3 antibody or an antigen binding fragment thereof;an anti-CTLA4 antibody or an antigen binding fragment thereof; ananti-HVEM antibody or an antigen binding fragment thereof; an anti-CD27antibody or an antigen binding fragment thereof; an anti-CD137 antibodyor an antigen binding fragment thereof; an anti-OX40 antibody or anantigen binding fragment thereof; an anti-CD28 antibody or an antigenbinding fragment thereof; an anti-PDL1 antibody or an antigen bindingfragment thereof; an anti-PDL2 antibody or an antigen binding fragmentthereof; an anti-GITR antibody or an antigen binding fragment thereof;an anti-ICOS antibody or an antigen binding fragment thereof; ananti-SIRPα antibody or an antigen binding fragment thereof; an anti-ILT2antibody or antigen binding fragment thereof; an anti-ILT3 antibody orantigen binding fragment thereof; an anti-ILT4 antibody or antigenbinding fragment thereof; an anti-ILT5 antibody or an antigen bindingfragment thereof; and an anti-4-1BB antibody or an antigen bindingfragment thereof. In one embodiment, the anti-PD1 antibody or an antigenbinding fragment thereof is selected from the group consisting of:pembrolizumab or an antigen binding fragment thereof and nivolumab or anantigen binding fragment thereof. In one embodiment, the anti-TIGITantibody or antigen binding fragment of the invention comprises: (i) aheavy chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 3, 79, 80, 81, 82,83 or 140; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO:4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO:5, 65, 66, 67, 68, 69,70, 71, 72, 73 or 141; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO:6, 74, 75, 76, 77, 78 or142. In one embodiment, the anti-TIGIT antibody or antigen bindingfragment thereof comprises: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO:88; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:89, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 134, 135 or 147; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 90; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:91; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 92, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122 or 148; and (vi) a light chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 93. In one embodiment, theanti-TIGIT antibody or antigen binding fragment of the inventioncomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3;(iv) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 6. In another embodiment, the anti-TIGIT antibody or antigen bindingfragment of the invention comprises: (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 57; (ii) a heavychain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 58; (iii) a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 59; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 60; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:61; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 62. In one embodiment, the anti-TIGITantibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 89, 134 or 135; (iii) a heavy chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) alight chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 91; (v) a light chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 92; and (vi) a light chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In oneembodiment, the anti-TIGIT antibody or antigen binding fragment thereofcomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 90; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 92; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 93.

The invention also provides a method of treating cancer in a subject inneed thereof, comprising administering to the subject an effectiveamount of an anti-TIGIT antibody or antigen binding fragment of theinvention, and further administering an anti-PD1 antibody or an antigenbinding fragment thereof. In one embodiment, the anti-PD1 antibody or anantigen binding fragment thereof is selected from the group consistingof: pembrolizumab or an antigen binding fragment thereof and nivolumabor an antigen binding fragment thereof. In one embodiment, theanti-TIGIT antibody or antigen binding fragment of the inventioncomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3,79, 80, 81, 82, 83 or 140; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO:4; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:5,65, 66, 67, 68, 69, 70, 71, 72, 73 or 141; and (vi) a light chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO:6,74, 75, 76, 77, 78 or 142. In one embodiment, the anti-TIGIT antibody orantigen binding fragment thereof comprises: (i) a heavy chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 88; (ii) aheavy chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 89, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 134, 135 or 147; (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) a lightchain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 91; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 92, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122 or 148; and (vi) a light chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 93. In one embodiment, theanti-TIGIT antibody or antigen binding fragment of the inventioncomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3;(iv) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 6. In another embodiment, the anti-TIGIT How antibody or antigenbinding fragment of the invention comprises: (i) a heavy chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 57; (ii) aheavy chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 58; (iii) a heavy chain variable region CDR3 comprising theamino acid sequence of SEQ ID NO: 59; (iv) a light chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 60; (v) a lightchain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 61; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 62. In one embodiment, the anti-TIGITantibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 89, 134 or 135; (iii) a heavy chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) alight chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 91; (v) a light chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 92; and (vi) a light chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In oneembodiment, the anti-TIGIT antibody or antigen binding fragment thereofcomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 90; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 92; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 93.

The invention also provides a method of treating an infection orinfectious disease in a subject, comprising administering to the subjectan effective amount of an antibody or antigen binding fragment of theinvention, optionally in association with a further therapeutic agent ortherapeutic procedure. In one embodiment, the subject been treated is ahuman subject. In one embodiment, the further therapeutic agent isselected from the group consisting of: an anti-PD1 antibody or anantigen binding fragment thereof; an anti-LAG3 antibody or an antigenbinding fragment thereof; an anti-VISTA antibody or an antigen bindingfragment thereof; an anti-BTLA antibody or an antigen binding fragmentthereof; an anti-TIM3 antibody or an antigen binding fragment thereof;an anti-CTLA4 antibody or an antigen binding fragment thereof; ananti-HVEM antibody or an antigen binding fragment thereof; an anti-CD27antibody or an antigen binding fragment thereof; an anti-CD137 antibodyor an antigen binding fragment thereof; an anti-OX40 antibody or anantigen binding fragment thereof; an anti-CD28 antibody or an antigenbinding fragment thereof; an anti-PDL1 antibody or an antigen bindingfragment thereof; an anti-PDL2 antibody or an antigen binding fragmentthereof; an anti-GITR antibody or an antigen binding fragment thereof;an anti-ICOS antibody or an antigen binding fragment thereof; ananti-SIRPα antibody or an antigen binding fragment thereof; an anti-ILT2antibody or antigen binding fragment thereof; an anti-ILT3 antibody orantigen binding fragment thereof; an anti-ILT4 antibody or antigenbinding fragment thereof; an anti-ILT5 antibody or an antigen bindingfragment thereof; and an anti-4-1BB antibody or an antigen bindingfragment thereof. In one embodiment, the anti-PD1 antibody or an antigenbinding fragment thereof; is selected from the group consisting of:pembrolizumab or an antigen binding fragment thereof and nivolumab or anantigen binding fragment thereof. In one embodiment, the anti-TIGITantibody or antigen binding fragment of the invention comprises: (i) aheavy chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 3, 79, 80, 81, 82,83 or 140; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO:4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO:5, 65, 66, 67, 68, 69,70, 71, 72, 73 or 141; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO:6, 74, 75, 76, 77, 78 or142. In one embodiment, the anti-TIGIT antibody or antigen bindingfragment thereof comprises: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO:88; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:89, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 134, 135 or 147; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 90; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:91; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 92, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122 or 148; and (vi) a light chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 93. In one embodiment, theanti-TIGIT antibody or antigen binding fragment of the inventioncomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3;(iv) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 6. In another embodiment, the anti-TIGIT antibody or antigen bindingfragment of the invention comprises: (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 57; (ii) a heavychain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 58; (iii) a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO:59; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 60; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:61; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 62. In one embodiment, the anti-TIGITantibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO:88; (ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 89, 134 or 135; (iii) a heavy chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) alight chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 91; (v) a light chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 92; and (vi) a light chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In oneembodiment, the anti-TIGIT antibody or antigen binding fragment thereofcomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 90; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 92; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 93.

The invention also provides a vaccine comprising an antibody or antigenbinding fragment of the invention. In one embodiment, the anti-TIGITantibody or antigen binding fragment of the invention comprises: (i) aheavy chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 3, 79, 80, 81, 82,83 or 1401; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5, 65, 66, 67, 68, 69,70, 71, 72, 73 or 141; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO:6, 74, 75, 76, 77, 78 or142. In one embodiment, the anti-TIGIT antibody or antigen bindingfragment thereof comprises: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:89, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 134, 135 or 147; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 90; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:91; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 92, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122 or 148; and (vi) a light chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 93. In one embodiment, theanti-TIGIT antibody or antigen binding fragment of the inventioncomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3;(iv) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 6. In another embodiment, the anti-TIGIT antibody or antigen bindingfragment of the invention comprises: (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 57; (ii) a heavychain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 58; (iii) a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 59; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 60; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:61; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 62. In one embodiment, the anti-TIGITantibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 89, 134 or 135; (iii) a heavy chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) alight chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 91; (v) a light chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 92; and (vi) a light chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In oneembodiment, the anti-TIGIT antibody or antigen binding fragment thereofcomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 90; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 92; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 93. In one embodiment, the vaccine further comprises an antigen.

The invention also provides a method for detecting the presence of aTIGIT peptide or a fragment thereof in a sample comprising contactingthe sample with an antibody or antigen binding fragment thereof of theinvention and detecting the presence of a complex between the antibodyor fragment and the peptide; wherein detection of the complex indicatesthe presence of the TIGIT peptide. In one embodiment, the anti-TIGITantibody or antigen binding fragment of the invention comprises: (i) aheavy chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO: 3, 79, 80, 81, 82,83 or 140; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO:4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO:5, 65, 66, 67, 68, 69,70, 71, 72, 73 or 141; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 6, 74, 75, 76, 77, 78or 142. In one embodiment, the antibody or antigen binding fragmentthereof comprises: (i) a heavy chain variable region CDR1 comprising theamino acid sequence of SEQ ID NO: 88; (ii) a heavy chain variable regionCDR2 comprising the amino acid sequence of SEQ ID NO: 89, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 134, 135or 147; (iii) a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 153; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 91; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:92, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 or 148; and(vi) a light chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 93. In one embodiment, the antibody or antigenbinding fragment of the invention comprises: (i) a heavy chain variableregion CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) aheavy chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising theamino acid sequence of SEQ ID NO: 3; (iv) a light chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a lightchain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 5; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 6. In another embodiment, the antibody orantigen binding fragment of the invention comprises: (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:57; (ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 58; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 59; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:60; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 61; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 62. In one embodiment,the antibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 89, 134 or 135; (iii) a heavy chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) alight chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 91; (v) a light chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 92; and (vi) a light chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In oneembodiment, the anti-TIGIT antibody or antigen binding fragment thereofcomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 90; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 92; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 93.

The invention also provides a method of increasing the activity of animmune cell, comprising contacting the immune cell with any one of theantibodies or antigen binding fragments of the invention. In oneembodiment, the invention provides a method of increasing the activityof an immune cell, comprising administering to a subject in need thereofan effective amount of an antibody or antigen binding fragments of theinvention. In one embodiment, the method is used for: the treatment ofcancer, the treatment of an infection or infectious disease, or as avaccine adjuvant. In one embodiment, the increase in activity of animmune cell can be detected by measuring the proliferation of the immunecell. For example, an increase in activity of a T cell can be detectedby measuring the proliferation of the T cell. In one embodiment, theincrease in activity of an immune cell can be detected by measuring Tcell activation ex vivo in a sample derived from the subject. In oneembodiment, the increase in T cell activity is determined by: (i)measuring mixed lymphocyte reactions or direct anti-CD3 mAb stimulationof T cell receptor (TCR) signaling to induce production of a cytokineselected from the group consisting of: IL-2, TNFα, IL-17, IFNγ, IL-1β,GM-CSF, RANTES, IL-6, IL-8, IL-5 and IL-13; (ii) measuring SEB inducedproduction of one or more cytokines selected from the group consistingof: IL-2, TNFα, IL-17, IFNγ, GM-CSF, RANTES, IL-6, IL-8, IL-5 and IL-13;or (iii) measuring TT induced production of a cytokine selected from thegroup consisting of: IL-2, TNFα, IL-17, IFNγ, GM-CSF, RANTES, IL-6,IL-8, IL-5 and IL-13. In one embodiment, the anti-TIGIT antibody orantigen binding fragment of the invention comprises: (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1;(ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 3, 79, 80, 81, 82, 83or 140; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5, 65, 66, 67, 68, 69,70, 71, 72, 73 or 141; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 6, 74, 75, 76, 77, 78or 142. In one embodiment, the anti-TIGIT antibody or antigen bindingfragment thereof comprises: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:89, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 134, 135 or 147; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 90; (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:91; (v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 92, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122 or 148; and (vi) a light chain variable region CDR3 comprisingthe amino acid sequence of SEQ ID NO: 93. In one embodiment, theanti-TIGIT antibody or antigen binding fragment of the inventioncomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3;(iv) a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO: 4; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 6. In another embodiment, the anti-TIGIT antibody or antigen bindingfragment of the invention comprises: (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 57; (ii) a heavychain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 58; (iii) a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 59; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 60; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:61; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 62. In one embodiment, the anti-TIGITantibody or antigen binding fragment thereof comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 88; (ii) a heavy chain variable region CDR2 comprising the aminoacid sequence of SEQ ID NO: 89, 134 or 135; (iii) a heavy chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 90; (iv) alight chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 91; (v) a light chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 92; and (vi) a light chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In oneembodiment, the anti-TIGIT antibody or antigen binding fragment thereofcomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 90; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 92; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 93.

The invention also comprises a method of treating cancer or infectiousdisease in a subject, comprising administering to the subject aneffective amount of an antagonist anti-TIGIT antibody and an antagonistanti-PD1 antibody, wherein the anti-TIGIT antibody has increasedeffector function when compared to a parental antibody. As used herein,a “parental anti-antibody” refers to antibody having a wild-type Fcregion and/or wild type glycosylation (i.e., glycosylation patternresulting from expression of the polypeptide in a non-engineeredmammalian host cell). The effector function of a parental antibody canbe increased by mutating its Fc region or by altering its glycosylation(as discussed in further detail below). In one embodiment, the anti-PD1antibody is administered prior to the administration of a parentalantibody. In one embodiment, the anti-PD1 antibody is administered 4-10days prior to the administration of the anti-TIGIT antibody. In oneembodiment, pretreatment treatment with anti-PD1 antibody may modulateimmune cells resulting in enhanced Fc-mediated function of theanti-TIGIT antibodies. In one embodiment, the anti-TIGIT antibodycomprises a human IgG1 constant domain. In one embodiment, treatmentwith the anti-TIGIT and anti-PD1 antibodies does not result in depletionof Tregs. In one embodiment, the anti-PD1 antibody or an antigen bindingfragment thereof; is selected from the group consisting of:pembrolizumab and nivolumab. In one embodiment, the anti-TIGIT antibodyor antigen binding fragment of the invention comprises: (i) a heavychain variable region CDR1 comprising the amino acid sequence of SEQ IDNO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 3 (iv) a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4;(v) a light chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment,the anti-TIGIT antibody or antigen binding fragment of the inventioncomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 89, 134 or 135; (iii) aheavy chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 90 (iv) a light chain variable region CDR1 comprising theamino acid sequence of SEQ ID NO: 91; (v) a light chain variable regionCDR2 comprising the amino acid sequence of SEQ ID NO: 92; and (vi) alight chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO: 93. In one embodiment, the anti-TIGIT antibody or antigenbinding fragment thereof comprises: (i) a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO: 88; (ii) a heavychain variable region CDR2 comprising the amino acid sequence of SEQ IDNO: 134; (iii) a heavy chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 90; (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 91; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:92; and (vi) a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO: 93.

The invention also comprises a method of treating cancer or infectiousdisease in a subject, comprising administering to the subject aneffective amount of an antagonist anti-TIGIT antibody and an antagonistanti-PD1 antibody, wherein the anti-TIGIT antibody is afucosylated. Inone embodiment, the anti-TIGIT antibody or antigen binding fragment ofthe invention comprises: (i) a heavy chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2;(iii) a heavy chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 3 (iv) a light chain variable region CDR1comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5;and (vi) a light chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO: 6. In one embodiment, the anti-TIGIT antibody orantigen binding fragment of the invention comprises: (i) a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:88; (ii) a heavy chain variable region CDR2 comprising the amino acidsequence of SEQ ID NO: 89, 134 or 135; (iii) a heavy chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 90 (iv) alight chain variable region CDR1 comprising the amino acid sequence ofSEQ ID NO: 91; (v) a light chain variable region CDR2 comprising theamino acid sequence of SEQ ID NO: 92; and (vi) a light chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO: 93. In oneembodiment, the anti-TIGIT antibody or antigen binding fragment thereofcomprises: (i) a heavy chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 88; (ii) a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 134; (iii) a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 90; (iv) a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO: 91; (v) a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO: 92; and (vi) a lightchain variable region CDR3 comprising the amino acid sequence of SEQ IDNO: 93.

The invention also provides a method of increasing the anti-tumoractivity of an anti-TIGIT antibody comprising: obtaining a parentalanti-TIGIT antibody and increasing the effector function of the parentalanti-TIGIT antibody; wherein the activity of the resulting anti-TIGITantibody is increased as compared to the parental anti-TIGIT antibody.As used herein, a “parental anti-antibody” refers to antibody having awild-type Fc region and/or wild type glycosylation (i.e., glycosylationpattern resulting from expression of the polypeptide in a non-engineeredmammalian host cell). The effector function of a parental antibody canbe increased by mutating its Fc region or by altering its glycosylation,for example by making the antibody afucosylated (as discussed in furtherdetail below). In one embodiment, the effector function of a parentalanti-TIGIT antibody is increased by making mutations in the Fc region ofthe parental anti-TIGIT antibody. In another embodiment, the effectorfunction of a parental anti-TIGIT antibody is increased by removing thefucose residues from the antibody, or expressing the antibody in a hostcell that has been genetically engineered to remove the activity of theenzyme that adds fucose to glycoproteins.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows binding of antibody 14A6 to human and rhesus TIGIT(expressed in CHO-K1 cells).

FIG. 2 shows binding of antibody 28H5 to human and rhesus TIGIT(expressed in CHO-K1 cells).

FIG. 3 shows that antibodies 14A6 and 28H5 block hCD155 interaction withhTIGIT as determined by a cell-based ELISA blocking assay.

FIG. 4 shows binding of antibody 31C6 to human and rhesus TIGIT(expressed in CHO-K1 cells), and also shows that antibody 31C6 blockshCD155 interaction with hTIGIT.

FIG. 5 shows the activity of antibodies 14A6 and 28H5 in an in vitro Tcell assay.

FIG. 6 shows the activity of antibodies 14A6 and 31C6 in vitro T cellassay.

FIG. 7 shows expression of TIGIT, CD226, CD155 and CD96 in primary humanT cell lines. At day 3, clone BC4-49 shows highest up-regulation ofTIGIT (negative) ad down-regulation of CD226 (positive)

FIGS. 8A and 8B show the activity of various anti-TIGIT antibodies in anin vitro T cell assay. It shows that MBS43 and 14A6, 37D10 and 28H5anti-TIGIT antibodies rescue IFNγ responses in primary human T cells.

FIGS. 9A-9D show the effect of concurrent administration of a ratanti-mouse TIGIT antibody (GIGD7) and an anti-mouse PD-1 antibodycompared to the monotherapy treatment arms on the anti-tumor response ofmice implanted with the CT26 cell line (n=10/group). Treatment wascommenced when tumors reached 75 mm3-115 mm3.

FIG. 10 shows the effect of Fc isotype on the anti-tumor activity of ananti-TIGIT antibody (18G10) in combination with an anti-PD-1 antibody inan animal tumor model.

FIGS. 11A-11C show the effect of Fc isotype on the anti-tumor activityof an anti-TIGIT antibody (18G10) in combination with an anti-PD-1antibody in an animal tumor model.

FIG. 12 shows the effect of Fc isotype on the anti-tumor activity of ananti-TIGIT antibody (11A11) in combination with an anti-PD-1 antibody inan animal tumor model.

FIGS. 13A-13C show the effect of Fc isotype on the anti-tumor activityof an anti-TIGIT antibody (11A11) in combination with an anti-PD-1antibody in an animal tumor model.

FIG. 14 shows a heat map indicating regions of human TIGIT which arestrongly or weakly protected from deuteration by the binding of the 14A6antibody. The amino acid sequence shown in the heatmap corresponds toSEQ ID NO:87.

FIG. 15 shows a heat map indicating regions of human TIGIT which arestrongly or weakly protected from deuteration by the binding of the 31C6antibody. The amino acid sequence shown in the heatmap corresponds toSEQ ID NO:87.

FIG. 16 shows the effect of various humanized clones of 31C6 as comparedto chimera and parental antibodies in an engineered T cell functionalassay.

FIG. 17 shows the effect of various humanized clones of 31C6 as comparedto chimera antibodies in a primary T cell functional assay.

DETAILED DESCRIPTION Abbreviations

Throughout the detailed description and examples of the invention thefollowing abbreviations will be used:

-   ADCC Antibody-dependent cellular cytotoxicity-   CDC Complement-dependent cytotoxicity-   CDR Complementarity determining region in the immunoglobulin    variable regions, defined using the Kabat numbering system-   CHO Chinese hamster ovary-   ELISA Enzyme-linked immunosorbant assay-   FR Antibody framework region: the immunoglobulin variable regions    excluding the CDR regions.-   HRP Horseradish peroxidase-   IFN interferon-   IC50 concentration resulting in 50% inhibition-   IgG Immunoglobulin G-   Kabat An immunoglobulin alignment and numbering system pioneered by    Elvin A. Kabat ((1991) Sequences of Proteins of Immunological    Interest, 5th Ed. Public Health Service, National Institutes of    Health, Bethesda, Md.)-   mAb or Mab or MAb Monoclonal antibody-   SEB Staphylococcus Enterotoxin B-   TT Tetanus toxoid-   V region The segment of IgG chains which is variable in sequence    between different antibodies. It extends to Kabat residue 109 in the    light chain and 113 in the heavy chain.-   VH Immunoglobulin heavy chain variable region-   VK Immunoglobulin kappa light chain variable region

DEFINITIONS

So that the invention may be more readily understood, certain technicaland scientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientificterms used herein have the meaning commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise.

“Administration” and “treatment,” as it applies to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contact of an exogenous pharmaceutical, therapeutic, diagnosticagent, or composition to the animal, human, subject, cell, tissue,organ, or biological fluid. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” and “treatment”also means in vitro and ex vivo treatments, e.g., of a cell, by areagent, diagnostic, binding compound, or by another cell.

“Treat” or “treating” means to administer a therapeutic agent, such as acomposition containing any of the antibodies or antigen-bindingfragments of the present invention, internally or externally to asubject or patient having one or more disease symptoms, or beingsuspected of having a disease, for which the agent has therapeuticactivity. Typically, the agent is administered in an amount effective toalleviate one or more disease symptoms in the treated subject orpopulation, whether by inducing the regression of or inhibiting theprogression of such symptom(s) by any clinically measurable degree. Theamount of a therapeutic agent that is effective to alleviate anyparticular disease symptom may vary according to factors such as thedisease state, age, and weight of the patient, and the ability of thedrug to elicit a desired response in the subject. Whether a diseasesymptom has been alleviated can be assessed by any clinical measurementtypically used by physicians or other skilled healthcare providers toassess the severity or progression status of that symptom.

TIGIT

The term TIGIT includes human TIGIT, cynomolgous monkey TIGIT and rhesusTIGIT as well as fragments thereof such as the mature fragment thereoflacking the signal peptide. In an embodiment of the invention, the aminoacid sequence of human TIGIT comprises the amino acid sequence disclosedin amino acid residues 25-244 of Genbank Accession Number NP_776160.2(SEQ ID NO: 31). (Amino acid residues 1-24 of SEQ ID NO:31 correspond toa leader peptide.)

In an embodiment of the invention, the amino acid sequence ofcynomolgous monkey, e.g., Macaca fascicularis TIGIT comprises the aminoacid sequence disclosed in (SEQ ID NO: 32); see also Genbank Accessionno. XP_005548157. The amino acid sequence of rhesus monkey TIGIT isidentical to the amino acid sequence of cynomolgous monkey TIGIT. (Aminoacid residues 1-24 of SEQ ID NO:32 correspond to a leader peptide.)

Anti-TIGIT Antibodies and Antigen-Binding Fragments Thereof

The present invention provides antibodies or antigen-binding fragmentsthereof that bind human TIGIT and uses of such antibodies or fragments.In some embodiments, the anti-TIGIT antibodies are isolated.

As used herein, an anti-TIGIT antibody or antigen-binding fragmentthereof refers to an antibody or antigen-binding fragment thereof thatspecifically binds to human TIGIT. An antibody or antigen-bindingfragment thereof that “specifically binds to human TIGIT” is an antibodyor antigen-binding fragment thereof that binds to human TIGIT with a KDof about 1 nM or a higher affinity (e.g., 1 nM-2 pM, 1 nM, 100 pM, 10 pMor 2 pM), but does not bind to other proteins lacking this sequence. Forexample, an antibody that “specifically binds” human TIGIT does not bindto human CD226, human CD155 and human CD112. As a further example, anantibody or antigen-binding fragment that specifically binds to humanTIGIT may bind to a FLAG®-tagged form of human TIGIT but will not bindto other FLAG®-tagged proteins that lack human TIGIT epitopes. In oneembodiment, the antibody of the invention which specifically binds tohuman TIGIT is also cross-reactive with cynomolgus and rhesus TIGIT. Asused herein “cross-reactivity” refers to the ability of an antibody toreact with a homologous protein from other species. Whether an antibodyspecifically binds to human TIGIT can be determined using any assayknown in the art. Examples of assays known in the art to determiningbinding affinity include surface plasmon resonance (e.g., BIACORE) or asimilar technique (e.g. KinExa or OCTET).

The present invention includes anti-TIGIT antibodies and methods of usethereof. As used herein, the term “antibody” refers to any form ofantibody that exhibits the desired biological activity. Thus, it is usedin the broadest sense and specifically covers, but is not limited to,monoclonal antibodies (including full length monoclonal antibodiescomprising two light chains and two heavy chains), polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies),humanized antibodies, fully human antibodies, chimeric antibodies andcamelized single domain antibodies.

The present invention includes non-human parental (e.g. mouse androdent) anti-TIGIT antibodies and antigen-binding fragments thereof andmethods of use thereof. These antibodies may be modified for an intendeduse, such as humanization of an antibody for use as a human therapeuticantibody or fragment.

The present invention includes anti-TIGIT antigen-binding fragments andmethods of use thereof. As used herein, unless otherwise indicated,“antibody fragment” or “antigen-binding fragment” refers toantigen-binding fragments of antibodies, i.e. antibody fragments thatretain the ability to bind specifically to the antigen bound by thefull-length antibody, e.g. fragments that retain one or more CDRregions. Examples of antigen-binding fragments include, but are notlimited to, Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linearantibodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies andmultispecific antibodies formed from antibody fragments.

The present invention includes anti-TIGIT Fab fragments and methods ofuse thereof. A “Fab fragment” is comprised of one light chain and theC_(H)1 and variable regions of one heavy chain. The heavy chain of a Fabmolecule cannot form a disulfide bond with another heavy chain molecule.An “Fab fragment” can be the product of papain cleavage of an antibody.

The present invention includes anti-TIGIT antibodies and antigen-bindingfragments thereof which comprise an Fc region and methods of usethereof. An “Fc” region contains two heavy chain fragments comprisingthe C_(H)1 and C_(H)2 domains of an antibody. The two heavy chainfragments are held together by two or more disulfide bonds and byhydrophobic interactions of the C_(H)3 domains.

The present invention includes anti-TIGIT Fab′ fragments and methods ofuse thereof. A “Fab′ fragment” contains one light chain and a portion orfragment of one heavy chain that contains the V_(H) domain and theC_(H)1 domain and also the region between the C_(H)1 and C_(H)2 domains,such that an interchain disulfide bond can be formed between the twoheavy chains of two Fab′ fragments to form a F(ab′)₂ molecule.

The present invention includes anti-TIGIT F(ab′)₂ fragments and methodsof use thereof. A “F(ab′)₂ fragment” contains two light chains and twoheavy chains containing a portion of the constant region between theC_(H1) and C_(H2) domains, such that an interchain disulfide bond isformed between the two heavy chains. A F(ab′)₂ fragment thus is composedof two Fab′ fragments that are held together by a disulfide bond betweenthe two heavy chains. An “F(ab′)₂ fragment” can be the product of pepsincleavage of an antibody.

The present invention includes anti-TIGIT Fv fragments and methods ofuse thereof. The “Fv region” comprises the variable regions from boththe heavy and light chains, but lacks the constant regions.

The present invention includes anti-TIGIT scFv fragments and methods ofuse thereof. The term “single-chain Fv” or “scFv” antibody refers toantibody fragments comprising the V_(H) and V_(L) domains of anantibody, wherein these domains are present in a single polypeptidechain. Generally, the Fv polypeptide further comprises a polypeptidelinker between the V_(H) and V_(L) domains which enables the scFv toform the desired structure for antigen-binding. For a review of scFv,see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.See also, International Patent Application Publication No. WO 88/01649and U.S. Pat. Nos. 4,946,778 and 5,260,203.

The present invention includes anti-TIGIT domain antibodies and methodsof use thereof. A “domain antibody” is an immunologically functionalimmunoglobulin fragment containing only the variable region of a heavychain or the variable region of a light chain. In some instances, two ormore V_(H) regions are covalently joined with a peptide linker to createa bivalent domain antibody. The two V_(H) regions of a bivalent domainantibody may target the same or different antigens.

The present invention includes anti-TIGIT bivalent antibodies andmethods of use thereof. A “bivalent antibody” comprises twoantigen-binding sites. In some instances, the two binding sites have thesame antigen specificities. However, bivalent antibodies may bebispecific (see below).

The present invention includes anti-TIGIT camelized single domainantibodies and methods of use thereof. In certain embodiments,antibodies herein also include camelized single domain antibodies. See,e.g., Muyldermans et al. (2001) Trends Biochem. Sci. 26:230; Reichmannet al. (1999) J. Immunol. Methods 231:25; WO 94/04678; WO 94/25591; U.S.Pat. No. 6,005,079). In one embodiment, the present invention providessingle domain antibodies comprising two V_(H) domains with modificationssuch that single domain antibodies are formed.

The present invention includes anti-TIGIT diabodies and methods of usethereof. As used herein, the term “diabodies” refers to small antibodyfragments with two antigen-binding sites, which fragments comprise aheavy chain variable domain (V_(H)) connected to a light chain variabledomain (V_(L)) in the same polypeptide chain (V_(H)—V_(L) orV_(L)—V_(H)). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described more fully in, e.g., EP404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci.USA 90: 6444-6448. For a review of engineered antibody variantsgenerally see Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

Typically, an antibody or antigen-binding fragment of the inventionwhich is modified in some way retains at least 10% of its bindingactivity (when compared to the parental antibody) when that activity isexpressed on a molar basis. Preferably, an antibody or antigen-bindingfragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95%or 100% or more of the TIGIT binding affinity as the parental antibody.It is also intended that an antibody or antigen-binding fragment of theinvention can include conservative or non-conservative amino acidsubstitutions (referred to as “conservative variants” or “functionconserved variants” of the antibody) that do not substantially alter itsbiologic activity.

The present invention includes isolated anti-TIGIT antibodies andantigen-binding fragments thereof and methods of use thereof “Isolated”antibodies or antigen-binding fragments thereof are at least partiallyfree of other biological molecules from the cells or cell cultures inwhich they are produced. Such biological molecules include nucleicacids, proteins, lipids, carbohydrates, or other material such ascellular debris and growth medium. An isolated antibody orantigen-binding fragment may further be at least partially free ofexpression system components such as biological molecules from a hostcell or of the growth medium thereof. Generally, the term “isolated” isnot intended to refer to a complete absence of such biological moleculesor to an absence of water, buffers, or salts or to components of apharmaceutical formulation that includes the antibodies or fragments.

The present invention includes monoclonal anti-TIGIT antibodies andantigen-binding fragments thereof as well as monoclonal compositionscomprising a plurality of isolated monoclonal antibodies. The term“monoclonal antibody”, as used herein, refers to a population ofsubstantially homogeneous antibodies, i.e., the antibody moleculescomprising the population are identical in amino acid sequence exceptfor possible naturally occurring mutations that may be present in minoramounts. In contrast, conventional (polyclonal) antibody preparationstypically include a multitude of different antibodies having differentamino acid sequences in their variable domains, particularly their CDRsthat are often specific for different epitopes. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al. (1975) Nature 256: 495, or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al. (1991)Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597,for example. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

The present invention includes anti-TIGIT chimeric antibodies (e.g.,human constant domain/mouse variable domain) and methods of use thereof.As used herein, a “chimeric antibody” is an antibody having the variabledomain from a first antibody and the constant domain from a secondantibody, where the first and second antibodies are from differentspecies. (U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc.Natl. Acad. Sci. USA 81: 6851-6855). Typically, the variable domains areobtained from an antibody from an experimental animal (the “parentalantibody”), such as a rodent, and the constant domain sequences areobtained from human antibodies, so that the resulting chimeric antibodywill be less likely to elicit an adverse immune response in a humansubject than the parental (e.g., mouse) antibody.

The present invention includes anti-TIGIT humanized antibodies andantigen-binding fragments thereof (e.g., rat or mouse antibodies thathave been humanized) and methods of use thereof. The invention includesany humanized version of the 14A6 antibody (comprising SEQ ID NOs:7 and8), the 28H5 antibody (comprising SEQ ID NOs:63 and 64) and the 31C6antibody (comprising SEQ ID NOs: 94-95). As used herein, the term“humanized antibody” refers to forms of antibodies that containsequences from both human and non-human (e.g., mouse or rat) antibodies.In general, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin, and all or substantially all of the framework(FR) regions are those of a human immunoglobulin sequence. The humanizedantibody may optionally comprise at least a portion of a humanimmunoglobulin constant region (Fc).

In general, the basic antibody structural unit comprises a tetramer.Each tetramer includes two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of the heavy chain maydefine a constant region primarily responsible for effector function.Typically, human light chains are classified as kappa and lambda lightchains. Furthermore, human heavy chains are typically classified as mu,delta, gamma, alpha, or epsilon, and define the antibody's isotype asIgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavychains, the variable and constant regions are joined by a “J” region ofabout 12 or more amino acids, with the heavy chain also including a “D”region of about 10 more amino acids. See generally, FundamentalImmunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, in general, an intact antibody has two bindingsites. Except in bifunctional or bispecific antibodies, the two bindingsites are, in general, the same.

Typically, the variable domains of both the heavy and light chainscomprise three hypervariable regions, also called complementaritydetermining regions (CDRs), located within relatively conservedframework regions (FR). The CDRs are usually aligned by the frameworkregions, enabling binding to a specific epitope. In general, fromN-terminal to C-terminal, both light and heavy chains variable domainscomprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment ofamino acids to each domain is, generally, in accordance with thedefinitions of Sequences of Proteins of Immunological Interest, Kabat,et al.; National Institutes of Health, Bethesda, Md.; 5^(th) ed.; NIHPubl. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat,et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) JMol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody or antigen-binding fragment thereof that areresponsible for antigen-binding. The hypervariable region comprisesamino acid residues from a “complementarity determining region” or “CDR”(i.e. CDRL1, CDRL2 and CDRL3 in the light chain variable domain andCDRH1, CDRH2 and CDRH3 in the heavy chain variable domain). See Kabat etal. (1991) Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(defining the CDR regions of an antibody by sequence); see also Chothiaand Lesk (1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions ofan antibody by structure). As used herein, the term “framework” or “FR”residues refers to those variable domain residues other than thehypervariable region residues defined herein as CDR residues.

“Isolated nucleic acid molecule” or “isolated polynucleotide” means aDNA or RNA of genomic, mRNA, cDNA, or synthetic origin or somecombination thereof which is not associated with all or a portion of apolynucleotide in which the isolated polynucleotide is found in nature,or is linked to a polynucleotide to which it is not linked in nature.For purposes of this disclosure, it should be understood that “a nucleicacid molecule comprising” a particular nucleotide sequence does notencompass intact chromosomes. Isolated nucleic acid molecules“comprising” specified nucleic acid sequences may include, in additionto the specified sequences, coding sequences for up to ten or even up totwenty or more other proteins or portions or fragments thereof, or mayinclude operably linked regulatory sequences that control expression ofthe coding region of the recited nucleic acid sequences, and/or mayinclude vector sequences.

The phrase “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to use promoters,polyadenylation signals, and enhancers.

A nucleic acid or polynucleotide is “operably linked” when it is placedinto a functional relationship with another nucleic acid sequence. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, but not always, “operably linked” means that the DNAsequences being linked are contiguous, and, in the case of a secretoryleader, contiguous and in reading phase. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that not all progeny willhave precisely identical DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, “germline sequence” refers to a sequence of unrearrangedimmunoglobulin DNA sequences. Any suitable source of unrearrangedimmunoglobulin sequences may be used. Human germline sequences may beobtained, for example, from JOINSOLVER germline databases on the websitefor the National Institute of Arthritis and Musculoskeletal and SkinDiseases of the United States National Institutes of Health. Mousegermline sequences may be obtained, for example, as described inGiudicelli et al. (2005) Nucleic Acids Res. 33:D256-D261.

Physical and Functional Properties of the Exemplary Anti-TIGITAntibodies

The present invention provides anti-TIGIT antibodies and antigen-bindingfragments thereof having specified structural and functional features,and methods of use of the antibodies or antigen-binding fragmentsthereof in the treatment or prevention of disease (e.g., cancer orinfectious disease).

An “anti-TIGIT antibody or antigen-binding fragment thereof of thepresent invention” includes: any antibody or antigen-binding fragmentthereof that is discussed herein (e.g., 14A6, 28H5, 31C6 or humanizedversions of the these antibodies disclosed in Table 4) or a variantthereof (e.g., sequence variant or functional variant); any antibody orantigen-binding fragment comprising any one or more of the CDRs setforth in Table 4; any antibody or antigen-binding fragment that binds tothe same epitope in human TIGIT as the antibodies discussed herein(e.g., 14A6, 28H5 or 31C6); and any antibody or antigen-binding fragmentthat cross-blocks (partially or fully) or is cross-blocked (partially orfully) by an antibody discussed herein (e.g., 14A6, 28H5 or 31C6) forTIGIT binding.

Cross-blocking antibodies and antigen-binding fragments thereof can beidentified based on their ability to cross-compete with an antibody ofthe invention in standard binding assays (e.g., BIACore, ELISA, flowcytometry). For example, standard ELISA assays can be used in which arecombinant TIGIT (e.g., human TIGIT) protein is immobilized on theplate, one of the antibodies is fluorescently labeled and the ability ofnon-labeled antibodies to compete off the binding of the labeledantibody is evaluated. Additionally or alternatively, BIAcore analysiscan be used to assess the ability of the antibodies to cross-compete.The ability of a test antibody to inhibit the binding of anotherantibody (for example, antibody 14A6 or 28H5 or 31C6) to TIGIT (e.g.,human TIGIT) demonstrates that the test antibody can compete withanother antibody (e.g., 14A6 or 28H5 or 31C6) for binding to TIGIT(e.g., human TIGIT) and thus, may, in some cases, bind to the sameepitope on TIGIT (e.g., human TIGIT) as antibody 14A6 or 28H5 or 31C6 orto an overlapping epitope.

As stated above, antibodies and fragments that bind to the same epitopeas any of the anti-TIGIT antibodies or antigen-binding fragments thereofof the present invention also form part of the present invention.Further, antibodies that bind to an epitope that overlaps with theepitope bound by any of the anti-TIGIT antibodies of the invention alsoform part of the present invention. There are several methods availablefor mapping antibody epitopes on target antigens, including: H/D-Ex Massspec, X-ray crystallography, pepscan analysis and site directedmutagenesis. For example, HDX (Hydrogen Deuterium Exchange) coupled withproteolysis and mass spectrometry can be used to determine the epitopeof an antibody on a specific antigen Y. HDX-MS relies on the accuratemeasurement and comparison of the degree of deuterium incorporation byan antigen when incubated in D₂O on its own and in presence of itsantibody at various time intervals. Deuterium is exchanged with hydrogenon the amide backbone of the proteins in exposed areas whereas regionsof the antigen bound to the antibody will be protected and will showless or no exchange after analysis by LC-MS/MS of proteolytic fragments.Example 9 exemplifies the use of HDX to map the epitope bound byantibody 14A6.

Examples of the immunoglobulin chains of anti-TIGIT antibodies of theinvention as well as their CDRs include, but are not limited thosedisclosed in Table 4 (SEQ ID NOs: 1-30, 37-52, 57-83 or 88-167). Thepresent invention includes any polypeptide comprising or consisting ofthe amino acid sequences of SEQ ID NOs: 1-30, 37-52, 57-83 or 88-167,and recombinant nucleotides encoding such polypeptides.

The scope of the present invention includes isolated anti-TIGITantibodies and antigen-binding fragments thereof (e.g., humanizedantibodies), comprising a variant of an immunoglobulin chain set forthherein, e.g., any of SEQ ID NOs: 7-30, 37-52, 63-64, 94-95 or 124-133;wherein the variant exhibits one or more of the following properties:(i) binds human TIGIT; (ii) cross-reacts with cynomolgous and rhesusTIGIT; (iii) blocks binding of human TIGIT to human CD155 and humanCD112; (iv) increases T cell activation; (v) stimulates antigen-specificT-cell production of IL-2 and IFNγ; (vi) blocks induction of T cellsuppression of activation induced by TIGIT ligation with cognate ligandsCD155 and CD112.

In other embodiments, the invention provides antibodies orantigen-binding fragment thereof that binds human TIGIT (e.g., humanizedantibodies) and has V_(L) domains and V_(H) domains with at least 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NOs:7-30, 37-52, 63-64, 94-95 or 124-133; wherein the variant exhibits thedesired binding and properties, e.g., (i) binds human TIGIT with a KDvalue of about 1×10⁻⁹ M to about 1×10⁻¹² M as determined by surfaceplasmon resonance (e.g., BIACORE) or a similar technique (e g KinExa orOCTET); (ii) cross-reacts with cynomolgous and rhesus TIGIT; (iii)blocks binding of human TIGIT to human CD155 and human CD112; (iv)increases T cell activation; (v) stimulates antigen-specific T-cellproduction of IL-2 and IFNγ; (vi) blocks induction of T cell suppressionof activation induced by TIGIT ligation with cognate ligands CD155 andCD112.

“Conservatively modified variants” or “conservative substitution” refersto substitutions of amino acids in a protein with other amino acidshaving similar characteristics (e.g. charge, side-chain size,hydrophobicity/hydrophilicity, backbone conformation and rigidity,etc.), such that the changes can frequently be made without altering thebiological activity of the protein. Those of skill in this art recognizethat, in general, single amino acid substitutions in non-essentialregions of a polypeptide do not substantially alter biological activity(see, e.g., Watson et al. (1987) Molecular Biology of the Gene, TheBenjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition,substitutions of structurally or functionally similar amino acids areless likely to disrupt biological activity. Exemplary conservativesubstitutions are set forth in Table 1.

TABLE 1 Exemplary Conservative Amino Acid Substitutions Original residueConservative substitution Ala (A) Gly; Ser Arg (R) Lys; His Asn (N) Gln;His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly(G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

Function-conservative variants of the antibodies of the invention arealso contemplated by the present invention. “Function-conservativevariants,” as used herein, refers to antibodies or fragments in whichone or more amino acid residues have been changed without altering adesired property, such an antigen affinity and/or specificity. Suchvariants include, but are not limited to, replacement of an amino acidwith one having similar properties, such as the conservative amino acidsubstitutions of Table 1. Also provided are isolated polypeptidescomprising the V_(L) domains of the anti-TIGIT antibodies of theinvention (e.g., SEQ ID NOs: 8, 25-30 and 48-52), and isolatedpolypeptides comprising the V_(H) domains (e.g., SEQ ID NOs: 7, 9-24 and37-47) of the anti-TIGIT antibodies of the invention having up to 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid substitutions.Also provided are isolated polypeptides comprising the V_(L) domains ofthe anti-TIGIT antibodies of the invention (e.g., SEQ ID NO:64) andisolated polypeptides comprising the V_(H) domains (e.g., SEQ ID NO:63)of the anti-TIGIT antibodies of the invention having up to 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more amino acid substitutions. Alsoprovided are isolated polypeptides comprising the V_(L) domains of theanti-TIGIT antibodies of the invention (e.g., SEQ ID NOs: 95 and130-133) and isolated polypeptides comprising the V_(H) domains (e.g.,SEQ ID NOs: 94 and 124-129) of the anti-TIGIT antibodies of theinvention having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or moreamino acid substitutions.

In another embodiment, provided is an antibody or antigen-bindingfragment thereof that binds TIGIT and has V_(L) domains and V_(H)domains with at least 99% 98%, 97%, 96%, 95%, 90%, 85%, 80% or 75%sequence identity to one or more of the V_(L) domains or V_(H) domainsdescribed herein, and exhibits specific binding to TIGIT. In anotherembodiment the binding antibody or antigen-binding fragment thereof ofthe present invention comprises V_(L) and V_(H) domains (with andwithout signal sequence) having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30 or more amino acid substitutions, and exhibits specific bindingto TIGIT.

Polynucleotides and Polypeptides

The present invention further comprises the polynucleotides encoding anyof the polypeptides or immunoglobulin chains of anti-TIGIT antibodiesand antigen-binding fragments thereof of the invention. For example, thepresent invention includes the polynucleotides encoding the amino acidsdescribed in SEQ ID NOs: 1-30, 37-52, 57-83 and 88-167, as well aspolynucleotides which hybridize thereto and, also, any polypeptideencoded by such a hybridizing polynucleotide. In one embodiment, theinvention comprises a nucleic acid sequence comprising or consistingessentially of SEQ ID NO:84 or SEQ ID NO:85.

In general, the polynucleotides hybridize under low, moderate or highstringency conditions, and encode antibodies or antigen-bindingfragments thereof that maintain the ability to bind to TIGIT (human,rhesus and/or cynomolgous monkey, e.g., Macaca fascicularis). A firstpolynucleotide molecule is “hybridizable” to a second polynucleotidemolecule when a single stranded form of the first polynucleotidemolecule can anneal to the second polynucleotide molecule under theappropriate conditions of temperature and solution ionic strength (seeSambrook, et al., supra). The conditions of temperature and ionicstrength determine the “stringency” of the hybridization. Typical lowstringency hybridization conditions include 55° C., 5×SSC, 0.1% SDS andno formamide; or 30% formamide, 5×SSC, 0.5% SDS at 42° C. Typicalmoderate stringency hybridization conditions are 40% formamide, with 5×or 6×SSC and 0.1% SDS at 42° C. High stringency hybridization conditionsare 50% formamide, 5× or 6×SSC at 42° C. or, optionally, at a highertemperature (e.g., 57° C., 59° C., 60° C., 62° C., 63° C., 65° C. or 68°C.). In general, SSC is 0.15M NaCl and 0.015M Na-citrate. Hybridizationrequires that the two polynucleotide contain complementary sequences,although, depending on the stringency of the hybridization, mismatchesbetween bases are possible. The appropriate stringency for hybridizingpolynucleotides depends on the length of the polynucleotides and thedegree of complementation, variables well known in the art. The greaterthe degree of similarity or homology between two nucleotide sequences,the higher the stringency under which the nucleic acids may hybridize.For hybrids of greater than 100 nucleotides in length, equations forcalculating the melting temperature have been derived (see Sambrook, etal., supra, 9.50-9.51). For hybridization with shorter polynucleotides,e.g., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook, et al., supra, 11.7-11.8).

In another embodiment, an isolated polynucleotide, for example DNA,encoding the polypeptide chains of the isolated antibodies orantigen-binding fragments set forth herein is provided. In oneembodiment, the isolated polynucleotide encodes an antibody orantigen-binding fragment thereof comprising at least one matureimmunoglobulin light chain variable (VL) domain according to theinvention and/or at least one mature immunoglobulin heavy chain variable(V_(H)) domain according to the invention. In some embodiments theisolated polynucleotide encodes both a light chain and a heavy chain ona single polynucleotide molecule, and in other embodiments the light andheavy chains are encoded on separate polynucleotide molecules. Inanother embodiment the polynucleotides further encodes a signalsequence.

In one embodiment, the invention comprises an isolated polynucleotideencoding an antibody heavy variable (V_(H)) domain or an antigen-bindingfragment thereof comprising CDR-H1 (SEQ ID NO:1), CDR-H2 (SEQ ID NO:2)and CDR-H3 (SEQ ID NO:3 or 79 or 80 or 81 or 82, 83 or 140).

In one embodiment, the invention comprises an isolated polynucleotideencoding an antibody light chain variable (V_(L)) domain or anantigen-binding fragment thereof comprising CDR-L1 (SEQ ID NO:4), CDR-L2(SEQ ID NO:5 or 65 or 66 or 67 or 68 or 69 or 70 or 71 or 72 or 73 or141) and CDR-L3 (SEQ ID NO:6 or 74 or 75 or 76 or 77 or 78).

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(H)) domain of SEQID NO: 7.

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(L)) domain of SEQID NO: 8.

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(H)) domain of anyone of SEQ ID NOs: 9-24 or 37-47.

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(L)) domain of anyone of SEQ ID NOs: 25-30 or 48-52

In one embodiment, the invention comprises an isolated polynucleotideencoding an antibody heavy variable (V_(H)) domain or an antigen-bindingfragment thereof comprising CDR-H1 (SEQ ID NO:57), CDR-H2 (SEQ ID NO:58)and CDR-H3 (SEQ ID NO:59).

In one embodiment, the invention comprises an isolated polynucleotideencoding an antibody light chain variable (V_(L)) domain or anantigen-binding fragment thereof comprising CDR-L1 (SEQ ID NO:60),CDR-L2 (SEQ ID NO:61) and CDR-L3 (SEQ ID NO:62).

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(H)) domain of SEQID NO: 63.

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(L)) domain of SEQID NO: 64.

In one embodiment, the invention comprises an isolated polynucleotideencoding an antibody heavy variable (V_(H)) domain or an antigen-bindingfragment thereof comprising CDR-H1 (SEQ ID NO: 88), CDR-H2 (SEQ ID NO:89, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 134 or 135) and CDR-H3 (SEQ ID NO: 90).

In one embodiment, the invention comprises an isolated polynucleotideencoding an antibody light chain variable (V_(L)) domain or anantigen-binding fragment thereof comprising CDR-L1 (SEQ ID NO: 91),CDR-L2 (SEQ ID NO: 92, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122 or 123) and CDR-L3 (SEQ ID NO: 93).

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(H)) domain of SEQID NO: 94.

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(L)) domain of SEQID NO: 95.

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(H)) domain of anyone of SEQ ID NOs: 124-129.

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(L)) domain of anyone of SEQ ID NOs: 130-133.

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(H)) domain of SEQID NO: 127.

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(H)) domain of SEQID NO: 128.

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(L)) domain of SEQID NO: 130.

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(L)) domain of SEQID NO: 132.

In one embodiment, the invention comprises an isolated polynucleotideencoding the immunoglobulin heavy chain variable (V_(L)) domain of SEQID NO: 133.

This present invention also provides vectors, e.g., expression vectors,such as plasmids, comprising the isolated polynucleotides of theinvention, wherein the polynucleotide is operably linked to controlsequences that are recognized by a host cell when the host cell istransfected with the vector. Also provided are host cells comprising avector of the present invention and methods for producing the antibodyor antigen-binding fragment thereof or polypeptide disclosed hereincomprising culturing a host cell harboring an expression vector or anucleic acid encoding the immunoglobulin chains of the antibody orantigen-binding fragment thereof in culture medium, and isolating theantigen or antigen-binding fragment thereof from the host cell orculture medium.

Also included in the present invention are polypeptides, e.g.,immunoglobulin polypeptides, comprising amino acid sequences that are atleast about 75% identical, 80% identical, more preferably at least about90% identical and most preferably at least about 95% identical (e.g.,95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequences of theantibodies provided herein when the comparison is performed by a BLASTalgorithm wherein the parameters of the algorithm are selected to givethe largest match between the respective sequences over the entirelength of the respective reference sequences (e.g. expect threshold: 10;word size: 3; max matches in a query range: 0; BLOSUM 62 matrix; gapcosts: existence 11, extension 1; conditional compositional score matrixadjustment).

Sequence identity refers to the degree to which the amino acids of twopolypeptides are the same at equivalent positions when the two sequencesare optimally aligned.

The following references relate to BLAST algorithms often used forsequence analysis: BLAST ALGORITHMS: Altschul et al. (2005) FEBS J.272(20): 5101-5109; Altschul, S. F., et al., (1990) J. Mol. Biol.215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden,T. L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S. F., etal., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997)Genome Res. 7:649-656; Wootton, J. C., et al., (1993) Comput. Chem.17:149-163; Hancock, J. M. et al., (1994) Comput. Appl. Biosci.10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al., “A model ofevolutionary change in proteins.” in Atlas of Protein Sequence andStructure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352,Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R. M., et al.,“Matrices for detecting distant relationships.” in Atlas of ProteinSequence and Structure, (1978) vol. 5, suppl. 3.” M. O. Dayhoff (ed.),pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.; Altschul, S.F., (1991) J. Mol. Biol. 219:555-565; States, D. J., et al., (1991)Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA89:10915-10919; Altschul, S. F., et al., (1993) J. Mol. Evol.36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl.Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob.22:2022-2039; and Altschul, S. F. “Evaluating the statisticalsignificance of multiple distinct local alignments.” in Theoretical andComputational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, N.Y.

Binding Affinity

By way of example, and not limitation, the antibodies andantigen-binding fragments disclosed herein may bind human TIGIT with aK_(D) value of at least about 1×10⁻⁹ M (i.e., a K_(D) value of 1×10⁻⁹ Mor lower) as determined by surface plasmon resonance (e.g., BIACORE) ora similar technique (e.g. KinExa or OCTET). In one embodiment, theantibodies and antigen-binding fragments disclosed herein may bind humanTIGIT with a K_(D) value of at least about 1×10⁻⁹ M to about 1×10⁻¹² Mas determined by surface plasmon resonance (e.g., BIACORE) or a similartechnique (e.g. KinExa or OCTET). In one embodiment, the antibodies andantigen-binding fragments disclosed herein may bind human TIGIT with aK_(D) value of at about 1×10⁻⁹ M to about 1×10⁻¹² M as determined bysurface plasmon resonance (e.g., BIACORE) or a similar technique (e.g.KinExa or OCTET). In one embodiment, the antibodies and antigen-bindingfragments disclosed herein may bind human TIGIT with a K_(D) value of atleast about 50 pM (i.e, a K_(D) value of about 50 pM or lower) asdetermined by BIACORE or a similar technique. In one embodiment, theantibodies and antigen-binding fragments disclosed herein may bind humanTIGIT with a K_(D) value of at least about 10 pM (i.e, a K_(D) value ofabout 10 pm lower) as determined by BIACORE or a similar technique. Inone embodiment, the antibodies and antigen-binding fragments of theinvention may bind to human TIGIT with a K_(D) of about 50 pM to about 1pM as determined by BIACORE or a similar technique.

Immune Cell Activation

In some embodiments, the antibodies or antigen binding fragments of theinvention increase the activity of an immune cell. The increase of theactivity of an immune cell can be detected using any method known in theart. In one embodiment, the increase in activity of an immune cell canbe detected by measuring the proliferation of the immune cell. Forexample, an increase in activity of a T cell can be detected bymeasuring the proliferation of the T cell or signal transduction eventssuch as tyrosine phosphorylation of immune receptors or downstreamkinases that transmit signals to transcriptional regulators. In otherembodiments, the increase in activity of an immune cell can be detectedby measuring CTL or NK cell cytotoxic function on specific target cellsor IFNγ cytokine responses, which are associated with stimulation ofanti-tumor immunity. In yet other embodiments, the increase in activityof an immune cell can be detected by measuring T cell activation ex vivoin a sample derived from the subject. In one embodiment, the increase inT cell activity is determined by: (i) measuring SEB (StaphylococcusEnterotoxin B) induced production of one or more pro-inflammatorycytokines selected from the group consisting of: IL-2, TNFα, IL-17,IFNγ, IL-1β, GM-CSF, RANTES, IL-6, IL-8, IL-5 and IL-13; or (ii)measuring mixed lymphocyte reactions or direct anti-CD3 mAb stimulationof T cell receptor (TCR) signaling to induce production of a cytokineselected from the group consisting of: IL-2, TNFα, IL-17, IFNγ, IL-1β,GM-CSF, RANTES, IL-6, IL-8, IL-5 and IL-13. In certain embodiments, theanti-TIGIT antibody or antigen binding fragment thereof of the presentinvention will stimulates antigen-specific T-cell production of IL-2and/or IFNγ by at least 1.5 fold.

The present invention includes antagonist anti-TIGIT antibodies andantigen-binding fragments thereof and methods of use thereof, e.g.,humanized, antagonist anti-TIGIT antibodies and fragments. An antagonistanti-TIGIT antibody or antigen-binding fragment thereof antagonizes anactivity of human TIGIT such as by inhibiting TIGIT binding to CD155 andCD112, and inhibiting functional ITIM signal transduction by TIGIT uponbinding to CD155 and CD112. Measurement of anti-TIGIT antagonistactivity can be assessed by demonstrating blocking of T cell suppressionfollowing TCR activation induced by TIGIT ligation with cognate ligandsCD155 and CD112. Hence, in one embodiment of increased responses,treating with antagonist anti-TIGIT antibodies are able to rescue IL-2responses to levels observed in T cells that are not repressed by CD155or CD112 induction of TIGIT. In a more preferred level of activation,responses, following treatment with an anti-TIGIT antagonist antibodymay increase responses to a level higher than T cell responses notrepressed by CD155 or CD112.

Ability of Anti-hTIGIT Antibodies to Block Binding to hCD155 and hCD112

In some embodiments, the anti-TIGIT antibodies or antigen bindingfragments of the invention are able to block binding of human TIGIT tohuman CD155 and human CD112. The ability to block binding of human TIGITto human CD155 and human CD112 can be determined using any method knownin the art. In one embodiment, the ability of the antibodies to blockbinding of human TIGIT to human CD155 and human CD112 is determinedusing an ELISA assay as described in Example 2.

Methods of Making Antibodies and Antigen-Binding Fragments Thereof

Hybridoma cells that produce parental (e.g., rat or mouse) monoclonalanti-TIGIT antibodies or antigen-binding fragments thereof discussedherein may be produced by methods which are commonly known in the art.Such isolated hybridomas are part of the present invention. Thesemethods include, but are not limited to, the hybridoma techniqueoriginally developed by Kohler, et al., (1975) (Nature 256:495-497), aswell as the trioma technique (Hering, et al., (1988) Biomed. Biochim.Acta. 47:211-216 and Hagiwara, et al., (1993) Hum. Antibod. Hybridomas4:15), the human B-cell hybridoma technique (Kozbor, et al., (1983)Immunology Today 4:72 and Cote, et al., (1983) Proc. Natl. Acad. Sci.U.S.A 80:2026-2030), the EBV-hybridoma technique (Cole, et al., inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96,1985), and electric field based electrofusion using a Cyto Pulse largechamber cull fusion electroporator (Cyto Pulse Sciences, Inc., GlenBurnie, Md.). Preferably, mouse splenocytes are isolated and fused withPEG or by electrofusion to a mouse myeloma cell line based upon standardprotocols. The resulting hybridomas may then be screened for theproduction of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice may by fused toone-sixth the number of P3×63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Cells may be plated at approximately2×10⁵ cells/mL in a flat bottom microtiter plate, followed by a two weekincubation in selective medium containing 20% fetal Clone Serum, 18%“653” conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mML-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50mg/ml gentamycin and 1×HAT (Sigma; the HAT is added 24 hours after thefusion). After two weeks, cells may be cultured in medium in which theHAT is replaced with HT. Individual wells may then be screened by ELISAfor anti-TIGIT monoclonal IgG antibodies. Once extensive hybridomagrowth occurs, medium can be observed usually after 10-14 days. Theantibody secreting hybridomas may be replated, screened again, and ifstill positive for human IgG, anti-TIGIT monoclonal antibodies, can besubcloned at least twice by limiting dilution. The stable subclones maythen be cultured in vitro to generate small amounts of antibody intissue culture medium for characterization.

Thus, the present invention includes methods for making an anti-TIGITantibody or antigen-binding fragment thereof of the present inventioncomprising culturing a hybridoma cell that expresses the antibody orfragment under condition favorable to such expression and, optionally,isolating the antibody or fragment from the hybridoma and/or the growthmedium (e.g. cell culture medium).

The anti-TIGIT antibodies disclosed herein may also be producedrecombinantly (e.g., in an E. coli/T7 expression system, a mammaliancell expression system or a lower eukaryote expression system). In thisembodiment, nucleic acids encoding the antibody immunoglobulin moleculesof the invention (e.g., V_(H) or V_(L)) may be inserted into a pET-basedplasmid and expressed in the E. coli/T7 system. For example, the presentinvention includes methods for expressing an antibody or antigen-bindingfragment thereof or immunoglobulin chain thereof in a host cell (e.g.,bacterial host cell such as E. coli such as BL21 or BL21DE3) comprisingexpressing T7 RNA polymerase in the cell which also includes apolynucleotide encoding an immunoglobulin chain that is operably linkedto a T7 promoter. For example, in an embodiment of the invention, abacterial host cell, such as a E. coli, includes a polynucleotideencoding the T7 RNA polymerase gene operably linked to a lac promoterand expression of the polymerase and the chain is induced by incubationof the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside).

There are several methods by which to produce recombinant antibodieswhich are known in the art. One example of a method for recombinantproduction of antibodies is disclosed in U.S. Pat. No. 4,816,567.

Transformation can be by any known method for introducingpolynucleotides into a host cell. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,biolistic injection and direct microinjection of the DNA into nuclei. Inaddition, nucleic acid molecules may be introduced into mammalian cellsby viral vectors. Methods of transforming cells are well known in theart. See, for example, U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461and 4,959,455.

Thus, the present invention includes recombinant methods for making ananti-TIGIT antibody or antigen-binding fragment thereof of the presentinvention, or an immunoglobulin chain thereof, comprising introducing apolynucleotide encoding one or more immunoglobulin chains of theantibody or fragment (e.g., heavy and/or light immunoglobulin chain);culturing the host cell (e.g., CHO or Pichia or Pichia pastoris) undercondition favorable to such expression and, optionally, isolating theantibody or fragment or chain from the host cell and/or medium in whichthe host cell is grown.

Anti-TIGIT antibodies can also be synthesized by any of the methods setforth in U.S. Pat. No. 6,331,415.

Eukaryotic and prokaryotic host cells, including mammalian cells ashosts for expression of the antibodies or fragments or immunoglobulinchains disclosed herein are well known in the art and include manyimmortalized cell lines available from the American Type CultureCollection (ATCC). These include, inter alia, Chinese hamster ovary(CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number ofother cell lines. Mammalian host cells include human, mouse, rat, dog,monkey, pig, goat, bovine, horse and hamster cells. Cell lines ofparticular preference are selected through determining which cell lineshave high expression levels. Other cell lines that may be used areinsect cell lines, such as Sf9 cells, amphibian cells, bacterial cells,plant cells and fungal cells. Fungal cells include yeast and filamentousfungus cells including, for example, Pichia pastoris, Pichia finlandica,Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichiaminuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorphs, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumlucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum,Physcomitrella patens and Neurospora crassa. Pichia sp., anySaccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candidaalbicans, any Aspergillus sp., Trichoderma reesei, Chrysosporiumlucknowense, any Fusarium sp., Yarrowia lipolytica, and Neurosporacrassa. When recombinant expression vectors encoding the heavy chain orantigen-binding portion or fragment thereof, the light chain and/orantigen-binding fragment thereof are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody orfragment or chain in the host cells or secretion of the into the culturemedium in which the host cells are grown.

Antibodies and antigen-binding fragments thereof and immunoglobulinchains can be recovered from the culture medium using standard proteinpurification methods. Further, expression of antibodies andantigen-binding fragments thereof and immunoglobulin chains of theinvention (or other moieties therefrom) from production cell lines canbe enhanced using a number of known techniques. For example, theglutamine synthetase gene expression system (the GS system) is a commonapproach for enhancing expression under certain conditions. The GSsystem is discussed in whole or part in connection with European PatentNos. 0 216 846, 0 256 055, and 0 323 997 and European Patent ApplicationNo. 89303964.4. Thus, in an embodiment of the invention, the mammalianhost cells (e.g., CHO) lack a glutamine synthetase gene and are grown inthe absence of glutamine in the medium wherein, however, thepolynucleotide encoding the immunoglobulin chain comprises a glutaminesynthetase gene which complements the lack of the gene in the host cell.

The present invention includes methods for purifying an anti-TIGITantibody or antigen-binding fragment thereof of the present inventioncomprising introducing a sample comprising the antibody or fragment to apurification medium (e.g., cation exchange medium, anion exchangemedium, hydrophobic exchange medium, affinity purification medium (e.g.,protein-A, protein-G, protein-A/G, protein-L)) and either collectingpurified antibody or fragment from the flow-through fraction of saidsample that does not bind to the medium; or, discarding the flow-throughfraction and eluting bound antibody or fragment from the medium andcollecting the eluate. In an embodiment of the invention, the medium isin a column to which the sample is applied. In an embodiment of theinvention, the purification method is conducted following recombinantexpression of the antibody or fragment in a host cell, e.g., wherein thehost cell is first lysed and, optionally, the lysate is purified ofinsoluble materials prior to purification on a medium.

In general, glycoproteins produced in a particular cell line ortransgenic animal will have a glycosylation pattern that ischaracteristic for glycoproteins produced in the cell line or transgenicanimal. Therefore, the particular glycosylation pattern of an antibodywill depend on the particular cell line or transgenic animal used toproduce the antibody. However, all antibodies encoded by the nucleicacid molecules provided herein, or comprising the amino acid sequencesprovided herein, comprise the instant invention, independent of theglycosylation pattern the antibodies may have. Similarly, in particularembodiments, antibodies with a glycosylation pattern comprising onlynon-fucosylated N-glycans may be advantageous, because these antibodieshave been shown to typically exhibit more potent efficacy than theirfucosylated counterparts both in vitro and in vivo (See for example,Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Pat. Nos.6,946,292 and 7,214,775). These antibodies with non-fucosylatedN-glycans are not likely to be immunogenic because their carbohydratestructures are a normal component of the population that exists in humanserum IgG.

The present invention includes polyclonal anti-TIGIT antibodies andantigen-binding fragments thereof, e.g., a composition comprising aplurality of anti-TIGIT antibodies and fragments, which include one ormore of the anti-TIGIT antibodies or antigen-binding fragments thereofof the present invention, and methods of use thereof. A polyclonalantibody is an antibody which was produced among or in the presence ofone or more other, non-identical antibodies. In general, polyclonalantibodies are produced from collections of different B-lymphocytes,e.g., the B-lymphocyte of an animal treated with an immunogen ofinterest, which produces a population of different antibodies but whichare all directed to the immunogen. Usually, polyclonal antibodies areobtained directly from an immunized animal, e.g., spleen, serum orascites fluid.

The present invention includes bispecific and bifunctional antibodiesand antigen-binding fragments having a binding specificity for TIGIT andanother antigen such as, for example, PD-1 or PD-L1 or LAG-3, andmethods of use thereof. In an embodiment of the invention, theanti-TIGIT chains comprise any one of the VH/VL sequences described inTable 4, and the PD1 chains comprise the amino acid sequence of SEQ IDNOs: 33 and 34 or of SEQ ID NOs: 35 and 36 (or an antigen bindingfragment of any of said sequences). A bispecific or bifunctionalantibody is an artificial hybrid antibody having two differentheavy/light chain pairs and two different binding sites. Bispecificantibodies can be produced by a variety of methods including fusion ofhybridomas or linking of Fab′ fragments. See, e.g., Songsivilai, et al.,(1990) Clin. Exp. Immunol. 79: 315-321, Kostelny, et al., (1992) JImmunol. 148:1547-1553. In addition, bispecific antibodies may be formedas “diabodies” (Holliger, et al., (1993) PNAS USA 90:6444-6448) or as“Janusins” (Traunecker, et al., (1991) EMBO J. 10:3655-3659 andTraunecker, et al., (1992) Int. J. Cancer Suppl. 7:51-52).

The present invention further includes anti-TIGIT antigen-bindingfragments of the anti-TIGIT antibodies disclosed herein. The antibodyfragments include F(ab)₂ fragments, which may be produced by enzymaticcleavage of an IgG by, for example, pepsin. Fab fragments may beproduced by, for example, reduction of F(ab)₂ with dithiothreitol ormercaptoethylamine.

Immunoglobulins may be assigned to different classes depending on theamino acid sequences of the constant domain of their heavy chains. Thereare at least five major classes of immunoglobulins: IgA, IgD, IgE, IgGand IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3 and IgG4; IgA1 and IgA2. The inventioncomprises antibodies and antigen-binding fragments of any of theseclasses or subclasses of antibodies.

In one embodiment, the antibody or antigen-binding fragment comprises aheavy chain constant region, e.g. a human constant region, such as γ1,γ2, γ3, or γ4 human heavy chain constant region or a variant thereof. Inanother embodiment, the antibody or antigen-binding fragment comprises alight chain constant region, e.g. a human light chain constant region,such as lambda or kappa human light chain region or variant thereof. Byway of example, and not limitation the human heavy chain constant regioncan be γ4 and the human light chain constant region can be kappa. In analternative embodiment, the Fc region of the antibody is γ4 with aSer228Pro mutation (Schuurman, J et. al., Mol. Immunol. 38: 1-8, 2001).

In one embodiment, the antibody or antigen-binding fragment comprises aheavy chain constant region of the IgG1 subtype.

In some embodiments, different constant domains may be appended tohumanized V_(L) and V_(H) regions derived from the CDRs provided herein.For example, if a particular intended use of an antibody (or fragment)of the present invention were to call for altered effector functions, aheavy chain constant domain other than human IgG1 may be used, or hybridIgG1/IgG4 may be utilized.

Although human IgG1 antibodies provide for long half-life and foreffector functions, such as complement activation and antibody-dependentcellular cytotoxicity, such activities may not be desirable for all usesof the antibody. In such instances a human IgG4 constant domain, forexample, may be used. The present invention includes anti-TIGITantibodies and antigen-binding fragments thereof which comprise an IgG4constant domain, e.g., antagonist, humanized anti-TIGIT antibodies andfragments, and methods of use thereof. In one embodiment, the IgG4constant domain can differ from the native human IgG4 constant domain(Swiss-Prot Accession No. P01861.1) at a position corresponding toposition 228 in the EU system and position 241 in the KABAT system,where the native Ser108 is replaced with Pro, in order to prevent apotential inter-chain disulfide bond between Cys106 and Cys109(corresponding to positions Cys 226 and Cys 229 in the EU system andpositions Cys 239 and Cys 242 in the KABAT system) that could interferewith proper intra-chain disulfide bond formation. See Angal et al.(1993) Mol. Imunol. 30:105. In other instances, a modified IgG1 constantdomain which has been modified to increase half-life or reduce effectorfunction can be used.

Antibody Engineering

Further included are embodiments in which the anti-TIGIT antibodies andantigen-binding fragments thereof are engineered antibodies to includemodifications to framework residues within the variable domains of aparental (e.g., mouse or rat) monoclonal antibody, e.g. to improve theproperties of the antibody or fragment. Typically, such frameworkmodifications are made to decrease the immunogenicity of the antibody orfragment. This is usually accomplished by replacing non-CDR residues inthe variable domains (i.e. framework residues) in a parental (e.g.rodent) antibody or fragment with analogous residues from the immunerepertoire of the species in which the antibody is to be used, e.g.human residues in the case of human therapeutics. Such an antibody orfragment is referred to as a “humanized” antibody or fragment. In somecases it is desirable to increase the affinity, or alter the specificityof an engineered (e.g. humanized) antibody. One approach is to“backmutate” one or more framework residues to the correspondinggermline sequence. More specifically, an antibody or fragment that hasundergone somatic mutation can contain framework residues that differfrom the germline sequence from which the antibody is derived. Suchresidues can be identified by comparing the antibody or fragmentframework sequences to the germline sequences from which the antibody orfragment is derived. Another approach is to revert to the originalparental (e.g., rodent) residue at one or more positions of theengineered (e.g. humanized) antibody, e.g. to restore binding affinitythat may have been lost in the process of replacing the frameworkresidues. (See, e.g., U.S. Pat. No. 5,693,762, U.S. Pat. No. 5,585,089and U.S. Pat. No. 5,530,101.)

In certain embodiments, the anti-TIGIT antibodies and antigen-bindingfragments thereof are engineered (e.g. humanized) to includemodifications to in the framework and/or CDRs to improve theirproperties. Such engineered changes can be based on molecular modelling.A molecular model for the variable region for the parental (non-human)antibody sequence can be constructed to understand the structuralfeatures of the antibody and used to identify potential regions on theantibody that can interact with the antigen. Conventional CDRs are basedon alignment of immunoglobulin sequences and identifying variableregions. Kabat et al., (1991) Sequences of Proteins of ImmunologicalInterest, Kabat, et al.; National Institutes of Health, Bethesda, Md.;5^(th) ed.; NIH Publ. No. 91-3242; Kabat (1978) Adv. Prot. Chem.32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616. Chothia andcoworkers carefully examined conformations of the loops in crystalstructures of antibodies and proposed hypervariable loops. Chothia, etal., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature342:878-883. There are variations between regions classified as “CDRs”and “hypervariable loops”. Later studies (Raghunathan et al, (2012) J.Mol Recog. 25, 3, 103-113) analyzed several antibody—antigen crystalcomplexes and observed that the antigen binding regions in antibodies donot necessarily conform strictly to the “CDR” residues or “hypervarible”loops. The molecular model for the variable region of the non-humanantibody can be used to guide the selection of regions that canpotentially bind to the antigen. In practice the potential antigenbinding regions based on model differ from the conventional “CDR”s or“hyper variable” loops. Commercial scientific software such as MOE(Chemical Computing Group) can be used for molecular modeling. Humanframeworks can be selected based on best matches with the non-humansequence both in the frameworks and in the CDRs. For FR4 (framework 4)in VH, VJ regions for the human germlines are compared with thecorresponding non-human region. In the case of FR4 (framework 4) in VL,J-kappa and J-Lambda regions of human germline sequences are comparedwith the corresponding non-human region. Once suitable human frameworksare identified, the CDRs are grafted into the selected human frameworks.In some cases certain residues in the VL-VH interface can be retained asin the non-human (parental) sequence. Molecular models can also be usedfor identifying residues that can potentially alter the CDRconformations and hence binding to antigen. In some cases, theseresidues are retained as in the non-human (parental) sequence. Molecularmodels can also be used to identify solvent exposed amino acids that canresult in unwanted effects such as glycosylation, deamidation andoxidation. Developability filters can be introduced early on in thedesign stage to eliminate/minimize these potential problems.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. Pat. No.7,125,689.

In particular embodiments, it will be desirable to change certain aminoacids containing exposed side-chains to another amino acid residue inorder to provide for greater chemical stability of the final antibody,so as to avoid deamidation or isomerization. The deamidation ofasparagine may occur on NG, DG, NG, NS, NA, NT, QG or QS sequences andresult in the creation of an isoaspartic acid residue that introduces akink into the polypeptide chain and decreases its stability (isoasparticacid effect). Isomerization can occur at DG, DS, DA or DT sequences. Incertain embodiments, the antibodies of the present disclosure do notcontain deamidation or asparagine isomerism sites.

For example, an asparagine (Asn) residue may be changed to Gln or Ala toreduce the potential for formation of isoaspartate at any Asn-Glysequences, particularly within a CDR. A similar problem may occur at aAsp-Gly sequence. Reissner and Aswad (2003) Cell. Mol. Life Sci.60:1281. Isoaspartate formation may debilitate or completely abrogatebinding of an antibody to its target antigen. See, Presta (2005) J.Allergy Clin. Immunol. 116:731 at 734. In one embodiment, the asparagineis changed to glutamine (Gln). It may also be desirable to alter anamino acid adjacent to an asparagine (Asn) or glutamine (Gln) residue toreduce the likelihood of deamidation, which occurs at greater rates whensmall amino acids occur adjacent to asparagine or glutamine. See,Bischoff & Kolbe (1994) J. Chromatog. 662:261. In addition, anymethionine residues (typically solvent exposed Met) in CDRs may bechanged to Lys, Leu, Ala, or Phe or other amino acids in order to reducethe possibility that the methionine sulfur would oxidize, which couldreduce antigen-binding affinity and also contribute to molecularheterogeneity in the final antibody preparation. Id. Additionally, inorder to prevent or minimize potential scissile Asn-Pro peptide bonds,it may be desirable to alter any Asn-Pro combinations found in a CDR toGln-Pro, Ala-Pro, or Asn-Ala. Antibodies with such substitutions aresubsequently screened to ensure that the substitutions do not decreasethe affinity or specificity of the antibody for TIGIT, or other desiredbiological activity to unacceptable levels.

TABLE 2 Exemplary stabilizing CDR variants CDR Residue StabilizingVariant Sequence Asn-Gly Gln-Gly, Ala-Gly, or Asn-Ala (N-G) (Q-G),(A-G), or (N-A) Asp-Gly Glu-Gly, Ala-Gly or Asp-Ala (D-G) (E-G), (A-G),or (D-A) Met (typically solvent exposed) Lys, Leu, Ala, or Phe (M) (K),(L), (A), or (F) Asn Gln or Ala (N) (Q) or (A) Asn-Pro Gin-Pro, Ala-Pro,or Asn-Ala (N-P) (Q-P), (A-P), or (N-A)

In some embodiments of the instant invention, the CDR3 of SEQ ID NO:3can be modified at position 110W to reduce or remove potential oxidation(wherein the numbering is according to Kabat). Thus, for example SEQ IDNO:3 (MPSFITLASLSTWEGYFDF) can be modified to any of the followingsequences: MPSFITLASLSTFEGYFDF (SEQ ID NO:79), MPSFITLASLSTYEGYFDF (SEQID NO:80), MPSFITLASLSTIEGYFDF (SEQ ID NO:81), MPSFITLASLSTVEGYFDF (SEQID NO:82) or MPSFITLASLSTLEGYFDF (SEQ ID NO:83). Therefore, in someembodiments of the instant invention, the anti-TIGIT antibody of theinvention comprises a heavy chain variable region comprising the CDR1 ofSEQ ID NO:1, the CDR2 of SEQ ID NO:2 and the CDR3 of SEQ ID NO: 3, 79,80, 81, 82 or 83.

In some embodiments of the instant invention, the CDR2 of SEQ ID NO:5can be modified at positions 52N and 53S to reduce or remove potentialdeamidation sites (wherein the numbering is according to Kabat). Thus,for example SEQ ID NO:5 (YANSLQT) can be modified to any of thefollowing sequences: YASNLQT (SEQ ID NO:65), YASSLQT (SEQ ID NO:66),YASTLQT (SEQ ID NO:67), YATTLQT (SEQ ID NO:68), YASYLQT (SEQ ID NO:69),YANQLQT (SEQ ID NO:70), YAGSLQT (SEQ ID NO:71), YASQLQT (SEQ ID NO:72),YADSLQT (SEQ ID NO:73). Therefore, in some embodiments of the instantinvention, the anti-TIGIT antibody of the invention comprises a lightchain variable region comprising the CDR1 of SEQ ID NO:4, the CDR2 ofSEQ ID NO:5, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQID NO:70, SEQ ID NO:72, SEQ ID NO:72 or SEQ ID NO:73, and the CDR3 ofSEQ ID NO:6.

In some embodiments of the instant invention, the CDR3 of SEQ ID NO:6can be modified at position 95W to reduce or remove potential oxidation(wherein the numbering is according to Kabat). Thus, for example SEQ IDNO:6 (QQYYSGWT) can be modified to any of the following sequences:QQYYSGFT (SEQ ID NO:74), QQYYSGYT (SEQ ID NO:75), QQYYSGIT (SEQ ID NO:76), QQYYSGVT (SEQ ID NO:77), QQYYSGLT (SEQ ID NO:78). Therefore, insome embodiments of the instant invention, the anti-TIGIT antibody ofthe invention comprises a light chain variable region comprising theCDR1 of SEQ ID NO:4, the CDR2 of SEQ ID NO:5 and the CDR3 of SEQ IDNO:6, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77 or SEQ IDNO:78.

In some embodiments of the instant invention, the anti-TIGIT antibody ofthe invention comprises a light chain variable region comprising theCDR1 of SEQ ID NO:4, the CDR2 of SEQ ID NO:5, SEQ ID NO:66, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:72 or SEQ ID NO:73, and the CDR3 of SEQ ID NO:6, SEQ ID NO:74, SEQ IDNO:75, SEQ ID NO:76, SEQ ID NO:77 or SEQ ID NO:78.

In another embodiment of the instant invention, the anti-TIGIT antibodyof the invention comprises a heavy chain FR4 region which comprises theamino acid sequence of any one of SEQ ID NOs: 7, 9-24 or 38-47, whereinthe M at position 122 is substituted with V, L, A, R, N, P Q, E, G, I,H, K, F, S, T, W, or L to avoid potential oxidation.

In another embodiment of the instant invention, the anti-TIGIT antibodyof the invention comprises a heavy chain FR4 region which comprises theamino acid sequence of any one of SEQ ID NOs: 7, 9-24 or 38-47, whereinthe M at position 122 and the V at position 123 are substituted with Tand L respectively to avoid potential oxidation.

In some embodiments of the instant invention, the CDR3 of SEQ ID NO:90can be modified at position 6 to reduce or remove potential oxidation.Thus, for example SEQ ID NO:90 (GGPYGWYFDV) be modified to any of thefollowing sequences: SEQ ID NOs: 154-167.

Antibody Engineering of the Fc Region

The antibodies (e.g., humanized antibodies) and antigen-bindingfragments thereof disclosed herein (e.g., 14A6, 28H5, 31C6 and humanizedversions thereof) can also be engineered to include modifications withinthe Fc region, typically to alter one or more properties of theantibody, such as serum half-life, complement fixation, Fc receptorbinding, and/or effector function (e.g., antigen-dependent cellularcytotoxicity). Furthermore, the antibodies and antigen-binding fragmentsthereof disclosed herein (e.g., 14A6, 28H5, 31C6 and humanized versionsthereof) can be chemically modified (e.g., one or more chemical moietiescan be attached to the antibody) or be modified to alter itsglycosylation, again to alter one or more properties of the antibody orfragment. Each of these embodiments is described in further detailbelow. The numbering of residues in the Fc region is that of the EUindex of Kabat.

The antibodies and antigen-binding fragments thereof disclosed herein(e.g., 14A6, 28H5, 31C6 and humanized versions thereof) also includeantibodies and fragments with modified (or blocked) Fc regions toprovide altered effector functions. See, e.g., U.S. Pat. No. 5,624,821;WO2003/086310; WO2005/120571; WO2006/0057702. Such modifications can beused to enhance or suppress various reactions of the immune system, withpossible beneficial effects in diagnosis and therapy. Alterations of theFc region include amino acid changes (substitutions, deletions andinsertions), glycosylation or deglycosylation, and adding multiple Fcregions. Changes to the Fc can also alter the half-life of antibodies intherapeutic antibodies, enabling less frequent dosing and thus increasedconvenience and decreased use of material. See Presta (2005) J. AllergyClin. Immunol. 116:731 at 734-35.

In one embodiment, the antibody or antigen-binding fragment of theinvention (e.g., 14A6, 28H5, 31C6 or humanized versions thereof) is anIgG4 isotype antibody or fragment comprising a Serine to Prolinemutation at a position corresponding to position 228 (S228P; EU index)in the hinge region of the heavy chain constant region. This mutationhas been reported to abolish the heterogeneity of inter-heavy chaindisulfide bridges in the hinge region (Angal et al. supra; position 241is based on the Kabat numbering system).

In one embodiment of the invention, the hinge region of CH1 is modifiedsuch that the number of cysteine residues in the hinge region isincreased or decreased. This approach is described further in U.S. Pat.No. 5,677,425. The number of cysteine residues in the hinge region ofCH1 is altered, for example, to facilitate assembly of the light andheavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody orantigen-binding fragment of the invention (e.g., 14A6, 28H5 or 31C6 orhumanized versions thereof) is mutated to decrease the biologicalhalf-life of the antibody or fragment. More specifically, one or moreamino acid mutations are introduced into the CH2-CH3 domain interfaceregion of the Fc-hinge fragment such that the antibody or fragment hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745.

In another embodiment, the antibody or antigen-binding fragment of theinvention (e.g., 14A6 or 28H5 or a humanized version thereof) ismodified to increase its biological half-life. Various approaches arepossible. For example, one or more of the following mutations can beintroduced: T252L, T254S, T256F, as described in U.S. Pat. No.6,277,375. Alternatively, to increase the biological half-life, theantibody can be altered within the CH1 or CL region to contain a salvagereceptor binding epitope taken from two loops of a CH2 domain of an Fcregion of an IgG, as described in U.S. Pat. Nos. 5,869,046 and6,121,022.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody or antigen-binding fragment. Forexample, one or more amino acids selected from amino acid residues 234,235, 236, 237, 297, 318, 320 and 322 can be replaced with a differentamino acid residue such that the antibody has an altered affinity for aneffector ligand and retains the antigen-binding ability of the parentantibody. The effector ligand to which affinity is altered can be, forexample, an Fc receptor or the C1 component of complement. This approachis described in further detail in U.S. Pat. Nos. 5,624,821 and5,648,260.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351.

In yet another example, the Fc region is modified to decrease theability of the antibody or antigen-binding fragment of the invention(e.g., 14A6 or 28H5 or a humanized version thereof) to mediate antibodydependent cellular cytotoxicity (ADCC) and/or to decrease the affinityof the antibody or fragment for an Fey receptor by modifying one or moreamino acids at the following positions: 238, 239, 243, 248, 249, 252,254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280,283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305,307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334,335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416,419, 430, 434, 435, 437, 438 or 439. This approach is described furtherin PCT Publication WO 00/42072. Moreover, the binding sites on humanIgG1 for FcγR1, FcγRII, FcγRIII and FcRn have been mapped and variantswith improved binding have been described (see Shields et al. (2001) J.Biol. Chem. 276:6591-6604).

In one embodiment of the invention, the Fc region is modified todecrease the ability of the antibody of the invention (e.g., 14A6, 28H5,31C6 and humanized versions thereof) to mediate effector function and/orto increase anti-inflammatory properties by modifying residues 243 and264. In one embodiment, the Fc region of the antibody or fragment ismodified by changing the residues at positions 243 and 264 to alanine.In one embodiment, the Fc region is modified to decrease the ability ofthe antibody or fragment to mediate effector function and/or to increaseanti-inflammatory properties by modifying residues 243, 264, 267 and328.

Effector Function Enhancement

In some embodiments, the Fc region of an anti-TIGIT antibody is modifiedto increase the ability of the antibody or antigen-binding fragment tomediate effector function and/or to increase their binding to theFcgamma receptors (FcγRs).

The term “Effector Function” as used herein is meant to refer to one ormore of Antibody Dependant Cell mediated Cytotoxic activity (ADCC),Complement-dependant cytotoxic activity (CDC) mediated responses,Fc-mediated phagocytosis or antibody dependant cellular phagocytosis(ADCP) and antibody recycling via the FcRn receptor.

The interaction between the constant region of an antigen bindingprotein and various Fc receptors (FCR) including FcgammaRI (CD64),FcgammaRII (CD32) and FcgammaRIII (CD16) is believed to mediate theeffector functions, such as ADCC and CDC, of the antigen bindingprotein. The Fc receptor is also important for antibody cross-linking,which can be important for anti-tumor immunity.

Effector function can be measured in a number of ways including forexample via binding of the FcgammaRIII to Natural Killer cells or viaFcgammaRI to monocytes/macrophages to measure for ADCC effectorfunction. For example an antigen binding protein of the presentinvention can be assessed for ADCC effector function in a Natural Killercell assay. Examples of such assays can be found in Shields et al, 2001J. Biol. Chem., Vol. 276, p 6591-6604; Chappel et al, 1993 J. Biol.Chem., Vol 268, p 25124-25131; Lazar et al, 2006 PNAS, 103; 4005-4010.

The ADCC or CDC properties of antibodies of the present invention, ortheir cross-linking properties, may be enhanced in a number of ways.

Human IgG1 constant regions containing specific mutations or alteredglycosylation on residue Asn297 have been shown to enhance binding to Fcreceptors. In some cases these mutations have also been shown to enhanceADCC and CDC (Lazar et al. PNAS 2006, 103; 4005-4010; Shields et al. JBiol Chem 2001, 276; 6591-6604; Nechansky et al. Mol Immunol, 2007, 44;1815-1817).

In one embodiment of the present invention, such mutations are in one ormore of positions selected from 239, 332 and 330 (IgG1), or theequivalent positions in other IgG isotypes. Examples of suitablemutations are S239D and I332E and A330L. In one embodiment, the antigenbinding protein of the invention herein described is mutated atpositions 239 and 332, for example S239D and I332E or in a furtherembodiment it is mutated at three or more positions selected from 239and 332 and 330, for example S239D and I332E and A330L. (EU indexnumbering).

In an alternative embodiment of the present invention, there is providedan antibody comprising a heavy chain constant region with an alteredglycosylation profile such that the antigen binding protein has enhancedeffector function. For example, wherein the antibody has enhanced ADCCor enhanced CDC or wherein it has both enhanced ADCC and CDC effectorfunction. Examples of suitable methodologies to produce antigen bindingproteins with an altered glycosylation profile are described inWO2003011878, WO2006014679 and EP1229125.

In a further aspect, the present invention provides “non-fucosylated” or“afucosylated” antibodies. Non-fucosylated antibodies harbour atri-mannosyl core structure of complex-type N-glycans of Fc withoutfucose residue. These glycoengineered antibodies that lack core fucoseresidue from the Fc N-glycans may exhibit stronger ADCC than fucosylatedequivalents due to enhancement of FcgammaRIIIa binding capacity.

The present invention also provides a method for the production of anantibody according to the invention comprising the steps of: a)culturing a recombinant host cell comprising an expression vectorcomprising the isolated nucleic acid as described herein, wherein therecombinant host cell does not comprise an alpha-1,6-fucosyltransferase;and b) recovering the antigen binding protein. The recombinant host cellmay be not normally contain a gene encoding analpha-1,6-fucosyltransferase (for example yeast host cells such asPichia sp.) or may have been genetically modified to inactive analpha-1,6-fucosyltransferase. Recombinant host cells which have beengenetically modified to inactivate the FUT8 gene encoding analpha-1,6-fucosyltransferase are available. See, e.g., the POTELLIGENT™technology system available from BioWa, Inc. (Princeton, N.J.) in whichCHOK1SV cells lacking a functional copy of the FUT8 gene producemonoclonal antibodies having enhanced antibody dependent cell mediatedcytotoxicity (ADCC) activity that is increased relative to an identicalmonoclonal antibody produced in a cell with a functional FUT8 gene.Aspects of the POTELLIGENT™ technology system are described in U.S. Pat.No. 7,214,775, U.S. Pat. No. 6,946,292, WO0061739 and WO0231240. Thoseof ordinary skill in the art will also recognize other appropriatesystems.

It will be apparent to those skilled in the art that such modificationsmay not only be used alone but may be used in combination with eachother in order to further enhance effector function.

Production of Antibodies with Modified Glycosylation

In still another embodiment, the antibodies or antigen-binding fragmentsof the invention (e.g., 14A6, 28H5, 31C6 and humanized versions thereof)comprise a particular glycosylation pattern. For example, anafucosylated or an aglycosylated antibody or fragment can be made (i.e.,the antibody lacks fucose or glycosylation, respectively). Theglycosylation pattern of an antibody or fragment may be altered to, forexample, increase the affinity or avidity of the antibody or fragmentfor a TIGIT antigen. Such modifications can be accomplished by, forexample, altering one or more of the glycosylation sites within theantibody or fragment sequence. For example, one or more amino acidsubstitutions can be made that result removal of one or more of thevariable region framework glycosylation sites to thereby eliminateglycosylation at that site. Such aglycosylation may increase theaffinity or avidity of the antibody or fragment for antigen. See, e.g.,U.S. Pat. Nos. 5,714,350 and 6,350,861.

Antibodies and antigen-binding fragments disclosed herein (e.g., 14A6,28H5, 31C6 and humanized versions thereof) may further include thoseproduced in lower eukaryote host cells, in particular fungal host cellssuch as yeast and filamentous fungi have been genetically engineered toproduce glycoproteins that have mammalian- or human-like glycosylationpatterns (See for example, Choi et al, (2003) Proc. Natl. Acad. Sci.100: 5022-5027; Hamilton et al., (2003) Science 301: 1244-1246; Hamiltonet al., (2006) Science 313: 1441-1443; Nett et al., Yeast 28(3):237-52(2011); Hamilton et al., Curr Opin Biotechnol. October; 18(5):387-92(2007)). A particular advantage of these genetically modified host cellsover currently used mammalian cell lines is the ability to control theglycosylation profile of glycoproteins that are produced in the cellssuch that compositions of glycoproteins can be produced wherein aparticular N-glycan structure predominates (see, e.g., U.S. Pat. No.7,029,872 and U.S. Pat. No. 7,449,308). These genetically modified hostcells have been used to produce antibodies that have predominantlyparticular N-glycan structures (See for example, Li et al., (2006) Nat.Biotechnol. 24: 210-215).

In particular embodiments, the antibodies and antigen-binding fragmentsthereof disclosed herein (e.g., 14A6, 28H5, 31C6 and humanized versionsthereof) further include those produced in lower eukaryotic host cellsand which comprise fucosylated and non-fucosylated hybrid and complexN-glycans, including bisected and multiantennary species, including butnot limited to N-glycans such as GlcNAc₍₁₋₄₎Man₃GlcNAc₂;Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂; NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂.

In particular embodiments, the antibodies and antigen-binding fragmentsthereof provided herein (e.g., 14A6, 28H5, 31C6 and humanized versionsthereof) may comprise antibodies or fragments having at least one hybridN-glycan selected from the group consisting of GlcNAcMan₅GlcNAc₂;GalGlcNAcMan₅GlcNAc₂; and NANAGalGlcNAcMan₅GlcNAc₂. In particularaspects, the hybrid N-glycan is the predominant N-glycan species in thecomposition.

In particular embodiments, the antibodies and antigen-binding fragmentsthereof provided herein (e.g., 14A6, 28H5, 31C6 and humanized versionsthereof) comprise antibodies and fragments having at least one complexN-glycan selected from the group consisting of GlcNAcMan₃GlcNAc₂;GalGlcNAcMan₃GlcNAc₂; NANAGalGlcNAcMan₃GlcNAc₂; GlcNAc₂Man₃GlcNAc₂;GalGlcNAc₂Man₃GlcNAc₂; Gal₂GlcNAc₂Man₃GlcNAc₂;NANAGal₂GlcNAc₂Man₃GlcNAc₂; and NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. Inparticular aspects, the complex N-glycan are the predominant N-glycanspecies in the composition. In further aspects, the complex N-glycan isa particular N-glycan species that comprises about 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycans inthe composition. In one embodiment, the antibody and antigen bindingfragments thereof provided herein comprise complex N-glycans, wherein atleast 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of thecomplex N-glycans in comprise the structure NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂,wherein such structure is afucosylated. Such structures can be produced,e.g., in engineered Pichia pastoris host cells.

In particular embodiments, the N-glycan is fucosylated. In general, thefucose is in an α1,3-linkage with the GlcNAc at the reducing end of theN-glycan, an α1,6-linkage with the GlcNAc at the reducing end of theN-glycan, an α1,2-linkage with the Gal at the non-reducing end of theN-glycan, an α1,3-linkage with the GlcNac at the non-reducing end of theN-glycan, or an α1,4-linkage with a GlcNAc at the non-reducing end ofthe N-glycan.

Therefore, in particular aspects of the above the glycoproteincompositions, the glycoform is in an α1,3-linkage or α1,6-linkage fucoseto produce a glycoform selected from the group consisting ofMan₅GlcNAc₂(Fuc), GlcNAcMan₅GlcNAc₂(Fuc), Man₃GlcNAc₂(Fuc),GlcNAcMan₃GlcNAc₂(Fuc), GlcNAc₂Man₃GlcNAc₂(Fuc),GalGlcNAc₂Man₃GlcNAc₂(Fuc), Gal₂GlcNAc₂Man₃GlcNAc₂(Fuc),NANAGal₂GlcNAc₂Man₃GlcNAc₂(Fuc), and NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂(Fuc);in an α1,3-linkage or α1,4-linkage fucose to produce a glycoformselected from the group consisting of GlcNAc(Fuc)Man₅GlcNAc₂,GlcNAc(Fuc)Man₃GlcNAc₂, GlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂,GalGlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂, Gal₂GlcNAc₂(Fuc1-2)Man3GlcNAc2,NANAGal2GlcNAc2(Fuc₁₋₂)Man₃GlcNAc₂, andNANA₂Gal₂GlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂; or in an α1,2-linkage fucose toproduce a glycoform selected from the group consisting ofGal(Fuc)GlcNAc₂Man₃GlcNAc₂, Gal₂(Fuc₁₋₂)GlcNAc₂Man₃GlcNAc₂,NANAGal₂(Fuc₁₋₂)GlcNAc₂Man₃GlcNAc₂, andNANA₂Gal₂(Fuc₁₋₂)GlcNAc₂Man₃GlcNAc₂.

In further aspects, the antibodies (e.g., humanized antibodies) orantigen-binding fragments thereof comprise high mannose N-glycans,including but not limited to, Man₈GlcNAc₂, Man₇GlcNAc₂, Man₆GlcNAc₂,Man₅GlcNAc₂, Man₄GlcNAc₂, or N-glycans that consist of the Man₃GlcNAc₂N-glycan structure.

In further aspects of the above, the complex N-glycans further includefucosylated and non-fucosylated bisected and multiantennary species.

As used herein, the terms “N-glycan” and “glycoform” are usedinterchangeably and refer to an N-linked oligosaccharide, for example,one that is attached by an asparagine-N-acetylglucosamine linkage to anasparagine residue of a polypeptide. N-linked glycoproteins contain anN-acetylglucosamine residue linked to the amide nitrogen of anasparagine residue in the protein. The predominant sugars found onglycoproteins are glucose, galactose, mannose, fucose,N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialicacid (e.g., N-acetyl-neuraminic acid (NANA)). The processing of thesugar groups occurs co-translationally in the lumen of the ER andcontinues post-translationally in the Golgi apparatus for N-linkedglycoproteins.

N-glycans have a common pentasaccharide core of Man₃GlcNAc₂ (“Man”refers to mannose; “Glc” refers to glucose; and “NAc” refers toN-acetyl; GlcNAc refers to N-acetylglucosamine). Usually, N-glycanstructures are presented with the non-reducing end to the left and thereducing end to the right. The reducing end of the N-glycan is the endthat is attached to the Asn residue comprising the glycosylation site onthe protein. N-glycans differ with respect to the number of branches(antennae) comprising peripheral sugars (e.g., GlcNAc, galactose, fucoseand sialic acid) that are added to the Man₃GlcNAc₂ (“Man3”) corestructure which is also referred to as the “trimannose core”, the“pentasaccharide core” or the “paucimannose core”. N-glycans areclassified according to their branched constituents (e.g., high mannose,complex or hybrid). A “high mannose” type N-glycan has five or moremannose residues. A “complex” type N-glycan typically has at least oneGlcNAc attached to the 1,3 mannose arm and at least one GlcNAc attachedto the 1,6 mannose arm of a “trimannose” core. Complex N-glycans mayalso have galactose (“Gal”) or N-acetylgalactosamine (“GalNAc”) residuesthat are optionally modified with sialic acid or derivatives (e.g.,“NANA” or “NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refersto acetyl). Complex N-glycans may also have intrachain substitutionscomprising “bisecting” GlcNAc and core fucose (“Fuc”). Complex N-glycansmay also have multiple antennae on the “trimannose core,” often referredto as “multiple antennary glycans.” A “hybrid” N-glycan has at least oneGlcNAc on the terminal of the 1,3 mannose arm of the trimannose core andzero or more mannoses on the 1,6 mannose arm of the trimannose core. Thevarious N-glycans are also referred to as “glycoforms.”

With respect to complex N-glycans, the terms “G-2”, “G-1”, “G0”, “G1”,“G2”, “A1”, and “A2” mean the following. “G-2” refers to an N-glycanstructure that can be characterized as Man₃GlcNAc₂; the term “G-1”refers to an N-glycan structure that can be characterized asGlcNAcMan₃GlcNAc₂; the term “G0” refers to an N-glycan structure thatcan be characterized as GlcNAc₂Man₃GlcNAc₂; the term “G1” refers to anN-glycan structure that can be characterized as GalGlcNAc₂Man₃GlcNAc₂;the term “G2” refers to an N-glycan structure that can be characterizedas Gal₂GlcNAc₂Man₃GlcNAc₂; the term “A1” refers to an N-glycan structurethat can be characterized as NANAGal₂GlcNAc₂Man₃GlcNAc₂; and, the term“A2” refers to an N-glycan structure that can be characterized asNANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. Unless otherwise indicated, the terms G-2″,“G-1”, “G0”, “G1”, “G2”, “A1”, and “A2” refer to N-glycan species thatlack fucose attached to the GlcNAc residue at the reducing end of theN-glycan. When the term includes an “F”, the “F” indicates that theN-glycan species contains a fucose residue on the GlcNAc residue at thereducing end of the N-glycan. For example, G0F, G1F, G2F, A1F, and A2Fall indicate that the N-glycan further includes a fucose residueattached to the GlcNAc residue at the reducing end of the N-glycan.Lower eukaryotes such as yeast and filamentous fungi do not normallyproduce N-glycans that produce fucose.

With respect to multiantennary N-glycans, the term “multiantennaryN-glycan” refers to N-glycans that further comprise a GlcNAc residue onthe mannose residue comprising the non-reducing end of the 1,6 arm orthe 1,3 arm of the N-glycan or a GlcNAc residue on each of the mannoseresidues comprising the non-reducing end of the 1,6 arm and the 1,3 armof the N-glycan. Thus, multiantennary N-glycans can be characterized bythe formulas GlcNAc₍₂₋₄₎Man₃GlcNAc₂, Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂, orNANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man3GlcNAc₂. The term “1-4” refers to 1, 2,3, or 4 residues.

With respect to bisected N-glycans, the term “bisected N-glycan” refersto N-glycans in which a GlcNAc residue is linked to the mannose residueat the reducing end of the N-glycan. A bisected N-glycan can becharacterized by the formula GlcNAc₃Man₃GlcNAc₂ wherein each mannoseresidue is linked at its non-reducing end to a GlcNAc residue. Incontrast, when a multiantennary N-glycan is characterized asGlcNAc₃Man₃GlcNAc₂, the formula indicates that two GlcNAc residues arelinked to the mannose residue at the non-reducing end of one of the twoarms of the N-glycans and one GlcNAc residue is linked to the mannoseresidue at the non-reducing end of the other arm of the N-glycan.

Antibody Physical Properties

The antibodies and antigen-binding fragments thereof disclosed herein(e.g., 14A6, 28H5, 31C6 and humanized versions thereof) may furthercontain one or more glycosylation sites in either the light or heavychain immunoglobulin variable region. Such glycosylation sites mayresult in increased immunogenicity of the antibody or fragment or analteration of the pK of the antibody due to altered antigen-binding(Marshall et al. (1972) Annu Rev Biochem 41:673-702; Gala and Morrison(2004) J Immunol 172:5489-94; Wallick et at (1988) J Exp Med168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et at (1985)Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).Glycosylation has been known to occur at motifs containing an N—X—S/Tsequence.

Each antibody or antigen-binding fragment (e.g., 14A6, 28H5, 31C6 orhumanized versions thereof) will have a unique isoelectric point (pI),which generally falls in the pH range between 6 and 9.5. The pI for anIgG1 antibody typically falls within the pH range of 7-9.5 and the pIfor an IgG4 antibody typically falls within the pH range of 6-8.

Each antibody or antigen-binding fragment (e.g., 14A6, 28H5, 31C6 orhumanized versions thereof) will have a characteristic meltingtemperature, with a higher melting temperature indicating greateroverall stability in vivo (Krishnamurthy R and Manning M C (2002) CurrPharm Biotechnol 3:361-71). In general, the T_(M1) (the temperature ofinitial unfolding) may be greater than 60° C., greater than 65° C., orgreater than 70° C. The melting point of an antibody or fragment can bemeasured using differential scanning calorimetry (Chen et at (2003)Pharm Res 20:1952-60; Ghirlando et at (1999) Immunol Lett 68:47-52) orcircular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).

In a further embodiment, antibodies and antigen-binding fragmentsthereof (e.g., 14A6, 28H5, 31C6 and humanized versions thereof) areselected that do not degrade rapidly. Degradation of an antibody orfragment can be measured using capillary electrophoresis (CE) andMALDI-MS (Alexander A J and Hughes D E (1995) Anal Chem 67:3626-32).

In a further embodiment, antibodies (e.g., 14A6, 28H5, 31C6 andhumanized versions thereof) and antigen-binding fragments thereof areselected that have minimal aggregation effects, which can lead to thetriggering of an unwanted immune response and/or altered or unfavorablepharmacokinetic properties. Generally, antibodies and fragments areacceptable with aggregation of 25% or less, 20% or less, 15% or less,10% or less, or 5% or less. Aggregation can be measured by severaltechniques, including size-exclusion column (SEC), high performanceliquid chromatography (HPLC), and light scattering.

Antibody Conjugates

The anti-TIGIT antibodies and antigen-binding fragments thereofdisclosed herein (e.g., 14A6, 28H5, 31C6 and humanized versions thereof)may also be conjugated to a chemical moiety. The chemical moiety may be,inter alia, a polymer, a radionuclide or a cytotoxic factor. Inparticular embodiments, the chemical moiety is a polymer which increasesthe half-life of the antibody or fragment in the body of a subject.Suitable polymers include, but are not limited to, hydrophilic polymerswhich include but are not limited to polyethylene glycol (PEG) (e.g.,PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30kDa or 40 kDa), dextran and monomethoxypolyethylene glycol (mPEG). Lee,et al., (1999) (Bioconj. Chem. 10:973-981) discloses PEG conjugatedsingle-chain antibodies. Wen, et al., (2001) (Bioconj. Chem. 12:545-553)disclose conjugating antibodies with PEG which is attached to aradiometal chelator (diethylenetriaminpentaacetic acid (DTPA)).

The antibodies and antigen-binding fragments thereof disclosed herein(e.g., 14A6, 28H5, 31C6 and humanized versions thereof) may also beconjugated with labels such as ⁹⁹Tc, ⁹⁰Y, ¹¹¹In, ³²P, ¹⁴C, ¹²⁵I, ³H,¹³¹I, ¹¹C, ¹⁵O, ¹³N, ¹⁸F, ³⁵S, ⁵¹Cr, ⁵⁷To, ²²⁶Ra, ⁶⁰Co, ⁵⁹Fe, ⁵⁷Se,¹⁵²Eu, ⁶⁷CU, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ²³⁴Th, and ⁴⁰K, ¹⁵⁷Gd,⁵⁵Mn, ⁵²Tr, and ⁵⁶Fe.

The antibodies and antigen-binding fragments disclosed herein (e.g.,14A6, 28H5, 31C6 and humanized versions thereof) may also be PEGylated,for example to increase its biological (e.g., serum) half-life. ToPEGylate an antibody or fragment, the antibody or fragment, typically isreacted with a reactive form of polyethylene glycol (PEG), such as areactive ester or aldehyde derivative of PEG, under conditions in whichone or more PEG groups become attached to the antibody or antibodyfragment. In particular embodiments, the PEGylation is carried out viaan acylation reaction or an alkylation reaction with a reactive PEGmolecule (or an analogous reactive water-soluble polymer). As usedherein, the term “polyethylene glycol” is intended to encompass any ofthe forms of PEG that have been used to derivatize other proteins, suchas mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethyleneglycol-maleimide. In certain embodiments, the antibody or fragment to bePEGylated is an aglycosylated antibody or fragment. Methods forPEGylating proteins are known in the art and can be applied to theantibodies of the invention. See, e.g., EP 0 154 316 and EP 0 401 384.

The antibodies and antigen-binding fragments disclosed herein (e.g.,14A6, 28H5, 31C6 and humanized versions thereof) may also be conjugatedwith fluorescent or chemilluminescent labels, including fluorophoressuch as rare earth chelates, fluorescein and its derivatives, rhodamineand its derivatives, isothiocyanate, phycoerythrin, phycocyanin,allophycocyanin, o-phthaladehyde, fluorescamine, ¹⁵²Eu, dansyl,umbelliferone, luciferin, luminal label, isoluminal label, an aromaticacridinium ester label, an imidazole label, an acridimium salt label, anoxalate ester label, an aequorin label, 2,3-dihydrophthalazinediones,biotin/avidin, spin labels and stable free radicals.

The antibodies and antigen-binding fragments thereof of the invention(e.g., 14A6, 28H5, 31C6 and humanized versions thereof) may also beconjugated to a cytotoxic factor such as diptheria toxin, Pseudomonasaeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin Achain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g.,fatty acids), dianthin proteins, Phytoiacca americana proteins PAPI,PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin,saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin,and enomycin.

Any method known in the art for conjugating the antibodies andantigen-binding fragments thereof of the invention (e.g., 14A6, 28H5,31C6 and humanized versions thereof) to the various moieties may beemployed, including those methods described by Hunter, et al., (1962)Nature 144:945; David, et al., (1974) Biochemistry 13:1014; Pain, etal., (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem,and Cytochem. 30:407. Methods for conjugating antibodies and fragmentsare conventional and very well known in the art.

Therapeutic Uses of Anti-TIGIT Antibodies

Further provided are methods for treating subjects, including humansubjects, in need of treatment with the isolated antibodies orantigen-binding fragments thereof disclosed herein (e.g., 14A6, 28H5,31C6 and humanized versions thereof). In one embodiment of theinvention, such subject suffers from an infection or an infectiousdisease. In another embodiment of the invention, such subject suffersfrom cancer. In one embodiment, the cancer is a solid tumor which isinfiltrated by tumor-infiltrating lymphocytes which express TIGIT. Inone embodiment the cancer is, e.g., osteosarcoma, rhabdomyosarcoma,neuroblastoma, kidney cancer, leukemia, renal transitional cell cancer,bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, breastcancer, prostate cancer, bone cancer, lung cancer (e.g., non-small celllung cancer), gastric cancer, colorectal cancer, cervical cancer,synovial sarcoma, head and neck cancer, squamous cell carcinoma,multiple myeloma, renal cell cancer, retinoblastoma, hepatoblastoma,hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney,Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma,pituitary adenoma, vestibular schwannoma, a primitive neuroectodermaltumor, medulloblastoma, astrocytoma, anaplastic astrocytoma,oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemiavera, thrombocythemia, idiopathic myelfibrosis, soft tissue sarcoma,thyroid cancer, endometrial cancer, carcinoid cancer or liver cancer,breast cancer or gastric cancer. In an embodiment of the invention, thecancer is metastatic cancer, e.g., of the varieties described above.

In an embodiment, the invention provides methods for treating subjectsusing an anti-TIGIT antibody or antigen-binding fragment thereof of theinvention (e.g., 14A6, 28H5, 31C6 and humanized versions thereof),wherein the subject suffers from a viral infection. In one embodiment,the viral infection is infection with a virus selected from the groupconsisting of human immunodeficiency virus (HIV), hepatitis virus (A, B,or C), herpes virus (e.g., VZV, HSV-I, HAV-6, HSV-II, and CMV, EpsteinBarr virus), adenovirus, influenza virus, flaviviruses, echovirus,rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus,mumps virus, rotavirus, measles virus, rubella virus, parvovirus,vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscumvirus, poliovirus, rabies virus, JC virus or arboviral encephalitisvirus.

In an embodiment, the invention provides methods for treating subjectsusing an anti-TIGIT antibody or antigen-binding fragment thereof of theinvention, wherein the subject suffers from a bacterial infection. Inone embodiment, the bacterial infection is infection with a bacteriaselected from the group consisting of Chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand gonococci, klebsiella, proteus, serratia, pseudomonas, Legionella,Corynebacterium diphtheriae, Salmonella, bacilli, Vibrio cholerae,Clostridium tetan, Clostridium botulinum, Bacillus anthricis, Yersiniapestis, Mycobacterium leprae, Mycobacterium lepromatosis, and Borriella.

In an embodiment, the invention provides methods for treating subjectsusing an anti-TIGIT antibody or antigen-binding fragment thereof of theinvention, wherein the subject suffers from a fungal infection. In oneembodiment, the fungal infection is infection with a fungus selectedfrom the group consisting of Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

In an embodiment, the invention provides methods for treating subjectsusing an anti-TIGIT antibody or antigen-binding fragment thereof of theinvention, wherein the subject suffers from a parasitic infection. Inone embodiment, the parasitic infection is infection with a parasiteselected from the group consisting of Entamoeba histolytica, Balantidiumcoli, Naegleria fowleri, Acanthamoeba, Giardia lambia, Cryptosporidium,Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosomabrucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii andNippostrongylus brasiliensis.

In addition, the present invention provides a method for preventing orinhibiting TIGIT binding to MHC class II, enhancing antigen-specificT-cell activation or stimulating T-cell production of interleukin-2 in asubject (e.g., human), for example, wherein the subject suffers fromcancer or infectious disease (e.g., as discussed herein) comprisingadministering an effective amount of anti-TIGIT antibody orantigen-binding fragment thereof (e.g., 14A6, 28A5, 31C6 and humanizedversions thereof), optionally in association with a furtherchemotherapeutic agent.

A “subject” may be a mammal such as a human, dog, cat, horse, cow,mouse, rat, monkey (e.g., cynomolgous monkey, e.g., Macaca fascicularis)or rabbit. In preferred embodiments of the invention, the subject is ahuman subject.

In particular embodiments, the antibodies or antigen-binding fragmentsthereof disclosed herein (e.g., 14A6, 28H5, 31C6 and humanized versionsthereof) may be used alone, or in association with other, furthertherapeutic agents and/or therapeutic procedures, for treating orpreventing any disease such as cancer, e.g., as discussed herein, in asubject in need of such treatment or prevention. Compositions, e.g.,pharmaceutical compositions comprising a pharmaceutically acceptablecarrier, comprising such antibodies and fragments in association withfurther therapeutic agents are also part of the present invention.

In particular embodiments, the antibodies or antigen-binding fragmentsthereof disclosed herein (e.g., 14A6, 28H5, 31C6 and humanized versionsthereof) may be used alone, or in association with tumor vaccines.

In particular embodiments, the antibodies or antigen-binding fragmentsthereof disclosed herein (e.g., 14A6, 28H5, 31C6 and humanized versionsthereof) may be used alone, or in association with chemotherapeuticagents.

In particular embodiments, the antibodies or antigen-binding fragmentsthereof disclosed herein (e.g., 14A6, 28H5, 31C6 and humanized versionsthereof) may be used alone, or in association with radiation therapy.

In particular embodiments, the antibodies or antigen-binding fragmentsthereof disclosed herein (e.g., 14A6, 28H5, 31C6 and humanized versionsthereof) may be used alone, or in association with targeted therapies.Examples of targeted therapies include: hormone therapies, signaltransduction inhibitors (e.g., EGFR inhibitors, such as cetuximab(Erbitux) and erlotinib (Tarceva)); HER2 inhibitors (e.g., trastuzumab(Herceptin) and pertuzumab (Perjeta)); BCR-ABL inhibitors (such asimatinib (Gleevec) and dasatinib (Sprycel)); ALK inhibitors (such ascrizotinib (Xalkori) and ceritinib (Zykadia)); BRAF inhibitors (such asvemurafenib (Zelboraf) and dabrafenib (Tafinlar)), gene expressionmodulators, apoptosis inducers (e.g., bortezomib (Velcade) andcarfilzomib (Kyprolis)), angiogenesis inhibitors (e.g., bevacizumab(Avastin) and ramucirumab (Cyramza), monoclonal antibodies attached totoxins (e.g., brentuximab vedotin (Adcetris) and ado-trastuzumabemtansine (Kadcyla)).

In particular embodiments, the anti-TIGIT antibodies or antigen-bindingfragments thereof of the invention (e.g., 14A6, 28H5, 31C6 and humanizedversions thereof) may be used in combination with an anti-cancertherapeutic agent or immunomodulatory drug such as an immunomodulatoryreceptor inhibitor, e.g., an antibody or antigen-binding fragmentthereof that specifically binds to the receptor.

Thus, the present invention includes compositions comprising ananti-TIGIT antibody or antigen-binding fragment thereof of the presentinvention (e.g., 14A6, 28H5, 31C6 and humanized versions thereof) inassociation with pembrolizumab; as well as methods for treating orpreventing cancer in a subject comprising administering an effectiveamount of the anti-TIGIT antibody or antigen-binding fragment thereofand pembrolizumab to the subject. Optionally, the subject is alsoadministered a further therapeutic agent.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the present invention (e.g., 14A6,28H5, 31C6 and humanized versions thereof) is in association with anisolated antibody comprising an immunoglobulin heavy chain comprisingthe amino acid sequence of SEQ ID NO:33 and an immunoglobulin lightchain comprising the amino acid sequence of SEQ ID NO:34. SEQ ID NOs: 33and 34 encode the heavy and light chain of pembrolizumab.

In an embodiment of the invention, an anti-TIGIT antibody) orantigen-binding fragment thereof of the present invention (e.g., 14A6,28H5, 31C6 and humanized versions thereof) is in association with anisolated antibody comprising an immunoglobulin heavy chain comprisingthe amino acid sequence of SEQ ID NO:35 and an immunoglobulin lightchain comprising the amino acid sequence of SEQ ID NO:36. SEQ ID NOs: 35and 36 encode the heavy and light chain of nivolumab.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 and humanized versions thereof) is in association with one or moreof: anti-PD1 antibody (e.g., pembrolizumab, nivolumab, pidilizumab(CT-011)), anti-PDLL antibody, anti-CTLA4 antibody, anti-CS1 antibody(e.g., elotuzumab), anti-KIR2DL1/2/3 antibody (e.g., lirilumab),anti-CD137 antibody (e.g., urelumab), anti-GITR antibody (e.g., TRX518),anti-PD-L1 antibody (e.g., BMS-936559, MSB0010718C or MPDL3280A),anti-PD-L2 antibody, anti-ILT1 antibody, anti-ILT2 antibody, anti-ILT3antibody, anti-ILT4 antibody, anti-ILT5 antibody, anti-ILT6 antibody,anti-ILT7 antibody, anti-ILT8 antibody, anti-CD40 antibody, anti-OX40antibody, anti-ICOS, anti-SIRPα, anti-KIR2DL1 antibody, anti-KIR2DL2/3antibody, anti-KIR2DL4 antibody, anti-KIR2DL5A antibody, anti-KIR2DL5Bantibody, anti-KIR3DL1 antibody, anti-KIR3DL2 antibody, anti-KIR3DL3antibody, anti-NKG2A antibody, anti-NKG2C antibody, anti-NKG2E antibody,anti-4-1BB antibody (e.g., PF-05082566), anti-TSLP antibody, anti-IL-10antibody, IL-10 or PEGylated IL-1β, or any small organic moleculeinhibitor of such targets.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-PD1antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-PDLLantibody (e.g., BMS-936559, MSB0010718C or MPDL3280A).

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-CTLA4antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-CS1antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with ananti-KIR2DL1/2/3 antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-CD137(e.g., urelumab) antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-GITR(e.g., TRX518) antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-PD-L2antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-ITL1antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-ITL2antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-ITL3antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-ITL4antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-ITL5antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-ITL6antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-ITL7antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-ITL8antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-CD40antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-OX40antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with ananti-KIR2DL1 antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with ananti-KIR2DL2/3 antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with ananti-KIR2DL4 antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with ananti-KIR2DL5A antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with ananti-KIR2DL5B antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with ananti-KIR3DL1 antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with ananti-KIR3DL2 antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with ananti-KIR3DL3 antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-NKG2Aantibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-NKG2Cantibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-ICOSantibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-SIRPαantibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-4-1BBantibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-IL-10antibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with an anti-TSLPantibody.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with IL-10 orPEGylated IL-10.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with one or moreof an inhibitor (e.g., a small organic molecule or an antibody orantigen-binding fragment thereof) such as: an MTOR (mammalian target ofrapamycin) inhibitor, a cytotoxic agent, a platinum agent, an EGFRinhibitor, a VEGF inhibitor, a microtubule stabilizer, a taxane, a CD20inhibitor, a CD52 inhibitor, a CD30 inhibitor, a RANK (Receptoractivator of nuclear factor kappa-B) inhibitor, a RANKL (Receptoractivator of nuclear factor kappa-B ligand) inhibitor, an ERK inhibitor,a MAP Kinase inhibitor, an AKT inhibitor, a MEK inhibitor, a PI3Kinhibitor, a HER1 inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4inhibitor, a Bcl2 inhibitor, a CD22 inhibitor, a CD79b inhibitor, anErbB2 inhibitor, or a farnesyl protein transferase inhibitor.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with any one ormore of: 13-cis-retinoic acid,3-[5-(methylsulfonylpiperadinemethyl)-indolyl]-quinolone,4-hydroxytamoxifen, 5-deooxyuridine, 5′-deoxy-5-fluorouridine,5-fluorouracil, 6-mecaptopurine, 7-hydroxystaurosporine, A-443654,abirateroneacetate, abraxane, ABT-578, acolbifene, ADS-100380, ALT-110,altretamine, amifostine, aminoglutethimide, amrubicin, Amsacrine,anagrelide, anastrozole, angiostatin, AP-23573, ARQ-197, arzoxifene,AS-252424, AS-605240, asparaginase, AT-9263, atrasentan, axitinib,AZD1152, Bacillus Calmette-Guerin (BCG) vaccine, batabulin, BC-210,besodutox, bevacizumab, bicalutamide, Bio111, BIO140, bleomycin,BMS-214662, BMS-247550, BMS-275291, BMS-310705, bortezimib, buserelin,busulfan, calcitriol, camptothecin, canertinib, capecitabine,carboplatin, carmustine, CC8490, Cediranib, CG-1521, CG-781,chlamydocin, chlorambucil, chlorotoxin, cilengitide, cimitidine,cisplatin, cladribine, clodronate, COL-3, CP-724714, cyclophosphamide,cyproterone, cyproteroneacetate, cytarabine, cytosinearabinoside,dacarbazine, dacinostat, dactinomycin, dalotuzumab, danusertib,dasatanib, daunorubicin, decatanib, deguelin, denileukin,deoxycoformycin, depsipeptide, diarylpropionitrile, diethylstilbestrol,diftitox, docetaxel, dovitinib, doxorubicin, droloxifene, edotecarin,yttrium-90 labeled-edotreotide, edotreotide, EKB-569, EMD121974,endostatin, enzalutamide, enzastaurin, epirubicin, epithilone B,ERA-923, Erbitux, erlotinib, estradiol, estramustine, etoposide,everolimus, exemestane, ficlatuzumab, finasteride, flavopiridol,floxuridine, fludarabine, fludrocortisone, fluoxymesterone, flutamide,FOLFOX regimen, Fulvestrant, galeterone, gefitinib, gemcitabine,gimatecan, goserelin, goserelin acetate, gossypol, GSK461364, GSK690693,HMR-3339, hydroxyprogesteronecaproate, hydroxyurea, IC87114, idarubicin,idoxyfene, ifosfamide, IM862, imatinib, IMC-1C11, INCB24360, INO1001,interferon, interleukin-12, ipilimumab, irinotecan, JNJ-16241199,ketoconazole, KRX-0402, lapatinib, lasofoxifene, letrozole, leucovorin,leuprolide, leuprolide acetate, levamisole, liposome entrappedpaclitaxel, lomustine, lonafarnib, lucanthone, LY292223, LY292696,LY293646, LY293684, LY294002, LY317615, marimastat, mechlorethamine,medroxyprogesteroneacetate, megestrolacetate, melphalan, mercaptopurine,mesna, methotrexate, mithramycin, mitomycin, mitotane, mitoxantrone,tozasertib, MLN8054, neovastat, Neratinib, neuradiab, nilotinib,nilutimide, nolatrexed, NVP-BEZ235, oblimersen, octreotide, ofatumumab,oregovomab, orteronel, oxaliplatin, paclitaxel, palbociclib,pamidronate, panitumumab, pazopanib, PD0325901, PD184352,PEG-interferon, pemetrexed, pentostatin, perifosine,phenylalaninemustard, PI-103, pictilisib, PIK-75, pipendoxifene,PKI-166, plicamycin, porfimer, prednisone, procarbazine, progestins,PX-866, R-763, raloxifene, raltitrexed, razoxin, ridaforolimus,rituximab, romidepsin, RTA744, rubitecan, scriptaid, Sdx102, seliciclib,selumetinib, semaxanib, SF1126, sirolimus, SN36093, sorafenib,spironolactone, squalamine, SR13668, streptozocin, SU6668,suberoylanalide hydroxamic acid, sunitinib, synthetic estrogen,talampanel, talimogene laherparepvec, tamoxifen, temozolomide,temsirolimus, teniposide, tesmilifene, testosterone, tetrandrine,TGX-221, thalidomide, thioguanine, thiotepa, ticilimumab, tipifarnib,tivozanib, TKI-258, TLK286, topotecan, toremifene citrate, trabectedin,trastuzumab, tretinoin, trichostatin A, triciribinephosphatemonohydrate, triptorelin pamoate, TSE-424, uracil mustard, valproicacid, valrubicin, vandetanib, vatalanib, VEGF trap, vinblastine,vincristine, vindesine, vinorelbine, vitaxin, vitespan, vorinostat,VX-745, wortmannin, Xr311, zanolimumab, ZK186619, ZK-304709, ZM336372,ZSTK474.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is in association with one or moreantiemetics including, but not limited to: casopitant (GlaxoSmithKline),Netupitant (MGI-Helsinn) and other NK-1 receptor antagonists,palonosetron (sold as Aloxi by MGI Pharma), aprepitant (sold as Emend byMerck and Co.; Rahway, N.J.), diphenhydramine (sold as Benadryl® byPfizer; New York, N.Y.), hydroxyzine (sold as Atarax® by Pfizer; NewYork, N.Y.), metoclopramide (sold as Reglan® by AH Robins Co.; Richmond,Va.), lorazepam (sold as Ativan® by Wyeth; Madison, N.J.), alprazolam(sold as Xanax® by Pfizer; New York, N.Y.), haloperidol (sold as Haldol®by Ortho-McNeil; Raritan, N.J.), droperidol (Inapsine®), dronabinol(sold as Marinol® by Solvay Pharmaceuticals, Inc.; Marietta, Ga.),dexamethasone (sold as Decadron® by Merck and Co.; Rahway, N.J.),methylprednisolone (sold as Medrol® by Pfizer; New York, N.Y.),prochlorperazine (sold as Compazine® by Glaxosmithkline; ResearchTriangle Park, N.C.), granisetron (sold as Kytril® by Hoffmann-La RocheInc.; Nutley, N.J.), ondansetron (sold as Zofran® by Glaxosmithkline;Research Triangle Park, N.C.), dolasetron (sold as Anzemet® bySanofi-Aventis; New York, N.Y.), tropisetron (sold as Navoban® byNovartis; East Hanover, N.J.).

Other side effects of cancer treatment include red and white blood celldeficiency. Accordingly, in an embodiment of the invention, ananti-TIGIT antibody or antigen-binding fragment thereof (e.g., 14A6,28H5, 31C6 or humanized versions thereof) is in association with anagent which treats or prevents such a deficiency, such as, e.g.,filgrastim, PEG-filgrastim, erythropoietin, epoetin alfa or darbepoetinalfa.

In an embodiment of the invention, an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6 or humanized versions thereof) is administered in association withanti-cancer radiation therapy. For example, in an embodiment of theinvention, the radiation therapy is external beam therapy (EBT): amethod for delivering a beam of high-energy X-rays to the location ofthe tumor. The beam is generated outside the patient (e.g., by a linearaccelerator) and is targeted at the tumor site. These X-rays can destroythe cancer cells and careful treatment planning allows the surroundingnormal tissues to be spared. No radioactive sources are placed insidethe patient's body. In an embodiment of the invention, the radiationtherapy is proton beam therapy: a type of conformal therapy thatbombards the diseased tissue with protons instead of X-rays. In anembodiment of the invention, the radiation therapy is conformal externalbeam radiation therapy: a procedure that uses advanced technology totailor the radiation therapy to an individual's body structures. In anembodiment of the invention, the radiation therapy is brachytherapy: thetemporary placement of radioactive materials within the body, usuallyemployed to give an extra dose—or boost—of radiation to an area.

In an embodiment of the invention, a surgical procedure administered inassociation with an anti-TIGIT antibody or antigen-binding fragmentthereof (e.g., 14A6, 28H5, 31C6 or humanized versions thereof) issurgical tumorectomy.

The term “in association with” indicates that the componentsadministered in a method of the present invention (e.g., an anti-TIGITantibody (e.g., humanized antibody) or antigen-binding fragment thereof(e.g., 14A6, 28H5, 31C6 or humanized versions thereof) along withpembrolizumab) can be formulated into a single composition forsimultaneous delivery or formulated separately into two or morecompositions (e.g., a kit). Each component can be administered to asubject at a different time than when the other component isadministered; for example, each administration may be givennon-simultaneously (e.g., separately or sequentially) at severalintervals over a given period of time. Moreover, the separate componentsmay be administered to a subject by the same or by a different route.

Experimental and Diagnostic Uses

The anti-TIGIT antibodies and antigen-binding fragments thereofdisclosed herein (e.g., 14A6, 28H5, 31C6 and humanized versions thereof)may be used as affinity purification agents. In this process, theanti-TIGIT antibodies and antigen-binding fragments thereof areimmobilized on a solid phase such a Sephadex, glass or agarose resin orfilter paper, using methods well known in the art. The immobilizedantibody or fragment is contacted with a sample containing the TIGITprotein (or a fragment thereof) to be purified, and thereafter thesupport is washed with a suitable solvent that will remove substantiallyall the material in the sample except the TIGIT protein, which is boundto the immobilized antibody or fragment. Finally, the support is washedwith a solvent which elutes the bound TIGIT (e.g., protein A). Suchimmobilized antibodies and fragments form part of the present invention.

Further provided are antigens for generating secondary antibodies whichare useful for example for performing Western blots and otherimmunoassays discussed herein. In particular, polypeptides are disclosedwhich comprise the variable regions and/or CDR sequences of atherapeutic antibody disclosed herein (e.g., 14A6, 28H5 or 31C6) andwhich may be used to generate anti-idiotypic antibodies for use inspecifically detecting the presence of the antibody, e.g., in atherapeutic context.

Anti-TIGIT antibodies (e.g., humanized antibodies) and antigen-bindingfragments thereof may also be useful in diagnostic assays for TIGITprotein, e.g., detecting its expression in specific cells, tissues, orserum, e.g., tumor cells such as melanoma cells. Such diagnostic methodsmay be useful in various disease diagnoses.

The present invention includes ELISA assays (enzyme-linked immunosorbentassay) incorporating the use of an anti-TIGIT antibody orantigen-binding fragment thereof disclosed herein (e.g., 14A6 or ahumanized version thereof).

For example, such a method comprises the following steps:

(a) coat a substrate (e.g., surface of a microtiter plate well, e.g., aplastic plate) with anti-TIGIT antibody or antigen-binding fragmentthereof;(b) apply a sample to be tested for the presence of TIGIT to thesubstrate;(c) wash the plate, so that unbound material in the sample is removed;(d) apply detectably labeled antibodies (e.g., enzyme-linked antibodies)which are also specific to the TIGIT antigen;(e) wash the substrate, so that the unbound, labeled antibodies areremoved;(f) if the labeled antibodies are enzyme linked, apply a chemical whichis converted by the enzyme into a fluorescent signal; and(g) detect the presence of the labeled antibody.

Detection of the label associated with the substrate indicates thepresence of the TIGIT protein.

In a further embodiment, the labeled antibody or antigen-bindingfragment thereof is labeled with peroxidase which react with ABTS (e.g.,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) or3,3′,5,5′-Tetramethylbenzidine to produce a color change which isdetectable. Alternatively, the labeled antibody or fragment is labeledwith a detectable radioisotope (e.g., ³H) which can be detected byscintillation counter in the presence of a scintillant.

An anti-TIGIT antibody or antigen-binding fragment thereof of theinvention (e.g., 14A6, 28H5, 31C6 or humanized versions thereof) may beused in a Western blot or immune-protein blot procedure. Such aprocedure forms part of the present invention and includes e.g.:

(1) optionally transferring proteins from a sample to be tested for thepresence of TIGIT (e.g., from a PAGE or SDS-PAGE electrophoreticseparation of the proteins in the sample) onto a membrane or other solidsubstrate using a method known in the art (e.g., semi-dry blotting ortank blotting); contacting the membrane or other solid substrate to betested for the presence of bound TIGIT or a fragment thereof with ananti-TIGIT antibody or antigen-binding fragment thereof of theinvention.

Such a membrane may take the form of a nitrocellulose or vinyl-based(e.g., polyvinylidene fluoride (PVDF)) membrane to which the proteins tobe tested for the presence of TIGIT in a non-denaturing PAGE(polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) gel have been transferred(e.g., following electrophoretic separation in the gel). Beforecontacting the membrane with the anti-TIGIT antibody or fragment, themembrane is optionally blocked, e.g., with non-fat dry milk or the likeso as to bind non-specific protein binding sites on the membrane.

(2) washing the membrane one or more times to remove unbound anti-TIGITantibody or fragment and other unbound substances; and(3) detecting the bound anti-TIGIT antibody or fragment.

Detection of the bound antibody or fragment indicates that the TIGITprotein is present on the membrane or substrate and in the sample.Detection of the bound antibody or fragment may be by binding theantibody or fragment with a secondary antibody (an anti-immunoglobulinantibody) which is detectably labeled and, then, detecting the presenceof the secondary antibody.

The anti-TIGIT antibodies and antigen-binding fragments thereofdisclosed herein (e.g., 14A6, 28H5, 31C6 and humanized versions thereof)may also be used for immunohistochemistry. Such a method forms part ofthe present invention and comprises, e.g.,

(1) contacting a cell (e.g., a tumor cell such as a melanoma cell) to betested for the presence of TIGIT protein with an anti-TIGIT antibody orantigen-binding fragment thereof of the invention; and(2) detecting the antibody or fragment on or in the cell.

If the antibody or fragment itself is detectably labeled, it can bedetected directly. Alternatively, the antibody or fragment may be boundby a detectably labeled secondary antibody which is detected.

Certain anti-TIGIT antibodies and antigen-binding fragments thereofdisclosed herein (e.g., 14A6, 28H5, 31C6 and humanized versions thereof)may also be used for in vivo tumor imaging. Such a method may includeinjection of a radiolabeled anti-TIGIT antibody or antigen-bindingfragment thereof into the body of a patient to be tested for thepresence of a tumor associated with TIGIT expression (e.g., whichexpresses TIGIT, for example, on the tumor cell surface) followed bynuclear imaging of the body of the patient to detect the presence of thelabeled antibody or fragment e.g., at loci comprising a highconcentration of the antibody or fragment which are bound to the tumor.The detection of the loci indicates the presence of the TIGIT⁺ tumor andtumor cells.

Imaging techniques include SPECT imaging (single photon emissioncomputed tomography) or PET imaging (positron emission tomography).Labels include e.g., iodine-123 (¹²³I) and technetium-99m (^(99m)Tc),e.g., in conjunction with SPECT imaging or ¹¹C, ¹³N, ¹⁵O or ¹⁸F, e.g.,in conjunction with PET imaging or Indium-111 (See e.g., Gordon et al.,(2005) International Rev. Neurobiol. 67:385-440).

Pharmaceutical Compositions and Administration

To prepare pharmaceutical or sterile compositions of the anti-TIGITantibodies and antigen-binding fragments of the invention (e.g., 14A6,28H5, 31C6 and humanized versions thereof), the antibody orantigen-binding fragment thereof is admixed with a pharmaceuticallyacceptable carrier or excipient. See, e.g., Remington's PharmaceuticalSciences and U.S. Pharmacopeia: National Formulary, Mack PublishingCompany, Easton, Pa. (1984).

Formulations of therapeutic and diagnostic agents may be prepared bymixing with acceptable carriers, excipients, or stabilizers in the formof, e.g., lyophilized powders, slurries, aqueous solutions orsuspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, N.Y.; Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Tablets, Marcel Dekker, N.Y.; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, N.Y.;Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, MarcelDekker, Inc., New York, N.Y.).

Toxicity and therapeutic efficacy of the antibodies of the invention,administered alone or in combination with another therapeutic agent, canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index (LD₅₀/ED₅₀). The data obtained fromthese cell culture assays and animal studies can be used in formulatinga range of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administration.

In a further embodiment, a further therapeutic agent that isadministered to a subject in association with an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., 14A6, 28H5,31C6) in accordance with the Physicians' Desk Reference 2003 (ThomsonHealthcare; 57th edition (Nov. 1, 2002)).

The mode of administration can vary. Routes of administration includeoral, rectal, transmucosal, intestinal, parenteral; intramuscular,subcutaneous, intradermal, intramedullary, intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, intraocular,inhalation, insufflation, topical, cutaneous, transdermal, orintra-arterial.

In particular embodiments, the anti-TIGIT antibodies or antigen-bindingfragments thereof of the invention (e.g., 14A6, 28H5, 31C6 and humanizedversions thereof) can be administered by an invasive route such as byinjection. In further embodiments of the invention, an anti-TIGITantibody or antigen-binding fragment thereof, or pharmaceuticalcomposition thereof, is administered intravenously, subcutaneously,intramuscularly, intraarterially, intratumorally, or by inhalation,aerosol delivery. Administration by non-invasive routes (e.g., orally;for example, in a pill, capsule or tablet) is also within the scope ofthe present invention.

The present invention provides a vessel (e.g., a plastic or glass vial,e.g., with a cap or a chromatography column, hollow bore needle or asyringe cylinder) comprising any of the antibodies or antigen-bindingfragments of the invention (e.g., 14A6, 28H5, 31C6 and humanizedversions thereof) or a pharmaceutical composition thereof. The presentinvention also provides an injection device comprising any of theantibodies or antigen-binding fragments of the invention (e.g., 14A6,28H5, 31C6 and humanized versions thereof) or a pharmaceuticalcomposition thereof. An injection device is a device that introduces asubstance into the body of a patient via a parenteral route, e.g.,intramuscular, subcutaneous or intravenous. For example, an injectiondevice may be a syringe (e.g., pre-filled with the pharmaceuticalcomposition, such as an auto-injector) which, for example, includes acylinder or barrel for holding fluid to be injected (e.g., antibody orfragment or a pharmaceutical composition thereof), a needle for piecingskin and/or blood vessels for injection of the fluid; and a plunger forpushing the fluid out of the cylinder and through the needle bore. In anembodiment of the invention, an injection device that comprises anantibody or antigen-binding fragment thereof of the present invention ora pharmaceutical composition thereof is an intravenous (IV) injectiondevice. Such a device includes the antibody or fragment or apharmaceutical composition thereof in a cannula or trocar/needle whichmay be attached to a tube which may be attached to a bag or reservoirfor holding fluid (e.g., saline; or lactated ringer solution comprisingNaCl, sodium lactate, KCl, CaCl₂ and optionally including glucose)introduced into the body of the patient through the cannula ortrocar/needle. The antibody or fragment or a pharmaceutical compositionthereof may, in an embodiment of the invention, be introduced into thedevice once the trocar and cannula are inserted into the vein of asubject and the trocar is removed from the inserted cannula. The IVdevice may, for example, be inserted into a peripheral vein (e.g., inthe hand or arm); the superior vena cava or inferior vena cava, orwithin the right atrium of the heart (e.g., a central IV); or into asubclavian, internal jugular, or a femoral vein and, for example,advanced toward the heart until it reaches the superior vena cava orright atrium (e.g., a central venous line). In an embodiment of theinvention, an injection device is an autoinjector; a jet injector or anexternal infusion pump. A jet injector uses a high-pressure narrow jetof liquid which penetrate the epidermis to introduce the antibody orfragment or a pharmaceutical composition thereof to a patient's body.External infusion pumps are medical devices that deliver the antibody orfragment or a pharmaceutical composition thereof into a patient's bodyin controlled amounts. External infusion pumps may be poweredelectrically or mechanically. Different pumps operate in different ways,for example, a syringe pump holds fluid in the reservoir of a syringe,and a moveable piston controls fluid delivery, an elastomeric pump holdsfluid in a stretchable balloon reservoir, and pressure from the elasticwalls of the balloon drives fluid delivery. In a peristaltic pump, a setof rollers pinches down on a length of flexible tubing, pushing fluidforward. In a multi-channel pump, fluids can be delivered from multiplereservoirs at multiple rates.

The pharmaceutical compositions disclosed herein may also beadministered with a needleless hypodermic injection device; such as thedevices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002; 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Suchneedleless devices comprising the pharmaceutical composition are alsopart of the present invention. The pharmaceutical compositions disclosedherein may also be administered by infusion. Examples of well-knownimplants and modules for administering the pharmaceutical compositionsinclude those disclosed in: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,447,233, which discloses a medicationinfusion pump for delivering medication at a precise infusion rate; U.S.Pat. No. 4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments. Many other such implants, delivery systems, and modulesare well known to those skilled in the art and those comprising thepharmaceutical compositions of the present invention are within thescope of the present invention.

Alternately, one may administer the anti-TIGIT antibody orantigen-binding fragment of the invention (e.g., 14A6, 28H5, 31C6 orhumanized versions thereof) in a local rather than systemic manner, forexample, via injection of the antibody or fragment directly into atumor, e.g., a TIGIT⁺ tumor. Furthermore, one may administer theantibody or fragment in a targeted drug delivery system, for example, ina liposome coated with a tissue-specific antibody, targeting, forexample, a tumor e.g., a TIGIT⁺ tumor, e.g., characterized byimmunopathology. The liposomes will be targeted to and taken upselectively by the afflicted tissue. Such methods and liposomes are partof the present invention.

The administration regimen depends on several factors, including theserum or tissue turnover rate of the therapeutic antibody orantigen-binding fragment, the level of symptoms, the immunogenicity ofthe therapeutic antibody, and the accessibility of the target cells inthe biological matrix. Preferably, the administration regimen deliverssufficient therapeutic antibody or fragment to effect improvement in thetarget disease state, while simultaneously minimizing undesired sideeffects. Accordingly, the amount of biologic delivered depends in parton the particular therapeutic antibody and the severity of the conditionbeing treated. Guidance in selecting appropriate doses of therapeuticantibodies or fragments is available (see, e.g., Wawrzynczak (1996)Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina(ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, MarcelDekker, New York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies andPeptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.;Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al.(1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl.J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med.342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky etal. (2000) New Engl. J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment. Generally, the dose begins with an amount somewhat less thanthe optimum dose and it is increased by small increments thereafteruntil the desired or optimum effect is achieved relative to any negativeside effects. Important diagnostic measures include those of symptomsof, e.g., the inflammation or level of inflammatory cytokines produced.In general, it is desirable that a biologic that will be used is derivedfrom the same species as the animal targeted for treatment, therebyminimizing any immune response to the reagent. In the case of humansubjects, for example, humanized and fully human antibodies are may bedesirable.

Antibodies or antigen-binding fragments thereof disclosed herein (e.g.,14A6, 28H5, 31C6 and humanized versions thereof) may be provided bycontinuous infusion, or by doses administered, e.g., daily, 1-7 timesper week, weekly, bi-weekly, monthly, bimonthly, quarterly,semiannually, annually etc. Doses may be provided, e.g., intravenously,subcutaneously, topically, orally, nasally, rectally, intramuscular,intracerebrally, intraspinally, or by inhalation. A total weekly dose isgenerally at least 0.05 μg/kg body weight, more generally at least 0.2μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.25 mg/kg, 1.0 mg/kg,2.0 mg/kg, 5.0 mg/ml, 10 mg/kg, 25 mg/kg, 50 mg/kg or more (see, e.g.,Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002)New Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J. Neurol.Neurosurg. Psych. 67:451-456; Portielji, et al. (20003) Cancer Immunol.Immunother. 52:151-144). Doses may also be provided to achieve apre-determined target concentration of anti-TIGIT antibody in thesubject's serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg/ml or more.In other embodiments, An anti-TIGIT antibody of the present invention isadministered, e.g., subcutaneously or intravenously, on a weekly,biweekly, “every 4 weeks,” monthly, bimonthly, or quarterly basis at 10,20, 50, 80, 100, 200, 500, 1000 or 2500 mg/subject.

As used herein, the term “effective amount” refer to an amount of ananti-TIGIT or antigen-binding fragment thereof of the invention (e.g.,humanized 14A6 or humanized 28H5) that, when administered alone or incombination with an additional therapeutic agent to a cell, tissue, orsubject, is effective to cause a measurable improvement in one or moresymptoms of disease, for example cancer or the progression of cancer. Aneffective dose further refers to that amount of the antibody or fragmentsufficient to result in at least partial amelioration of symptoms, e.g.,tumor shrinkage or elimination, lack of tumor growth, increased survivaltime. When applied to an individual active ingredient administeredalone, an effective dose refers to that ingredient alone. When appliedto a combination, an effective dose refers to combined amounts of theactive ingredients that result in the therapeutic effect, whetheradministered in combination, serially or simultaneously. An effectiveamount of a therapeutic will result in an improvement of a diagnosticmeasure or parameter by at least 10%; usually by at least 20%;preferably at least about 30%; more preferably at least 40%, and mostpreferably by at least 50%. An effective amount can also result in animprovement in a subjective measure in cases where subjective measuresare used to assess disease severity.

Kits

Further provided are kits comprising one or more components thatinclude, but are not limited to, an anti-TIGIT antibody orantigen-binding fragment, as discussed herein (e.g., humanized 14A6 orhumanized 28H5 or humanized 31C6) in association with one or moreadditional components including, but not limited to a pharmaceuticallyacceptable carrier and/or a therapeutic agent, as discussed herein. Theantibody or fragment and/or the therapeutic agent can be formulated as apure composition or in combination with a pharmaceutically acceptablecarrier, in a pharmaceutical composition.

In one embodiment, the kit includes an anti-TIGIT antibody orantigen-binding fragment thereof of the invention (e.g., humanized 14A6or humanized 28H5 or humanized 31C6) or a pharmaceutical compositionthereof in one container (e.g., in a sterile glass or plastic vial) anda pharmaceutical composition thereof and/or a therapeutic agent inanother container (e.g., in a sterile glass or plastic vial).

In another embodiment, the kit comprises a combination of the invention,including an anti-TIGIT antibody or antigen-binding fragment thereof ofthe invention (e.g., humanized 14A6 or humanized 28H5 or humanized 31C6)along with a pharmaceutically acceptable carrier, optionally incombination with one or more therapeutic agents formulated together,optionally, in a pharmaceutical composition, in a single, commoncontainer.

If the kit includes a pharmaceutical composition for parenteraladministration to a subject, the kit can include a device for performingsuch administration. For example, the kit can include one or morehypodermic needles or other injection devices as discussed above.

The kit can include a package insert including information concerningthe pharmaceutical compositions and dosage forms in the kit. Generally,such information aids patients and physicians in using the enclosedpharmaceutical compositions and dosage forms effectively and safely. Forexample, the following information regarding a combination of theinvention may be supplied in the insert: pharmacokinetics,pharmacodynamics, clinical studies, efficacy parameters, indications andusage, contraindications, warnings, precautions, adverse reactions,overdosage, proper dosage and administration, how supplied, properstorage conditions, references, manufacturer/distributor information andpatent information.

Detection Kits and Therapeutic Kits

As a matter of convenience, an anti-TIGIT antibody or antigen-bindingfragment thereof of the invention (e.g., 14A6, 28H5, 31C6 or humanizedversions thereof) can be provided in a kit, i.e., a packaged combinationof reagents in predetermined amounts with instructions for performingthe diagnostic or detection assay. Where the antibody or fragment islabeled with an enzyme, the kit will include substrates and cofactorsrequired by the enzyme (e.g., a substrate precursor which provides thedetectable chromophore or fluorophore). In addition, other additives maybe included such as stabilizers, buffers (e.g., a block buffer or lysisbuffer) and the like. The relative amounts of the various reagents maybe varied widely to provide for concentrations in solution of thereagents which substantially optimize the sensitivity of the assay.Particularly, the reagents may be provided as dry powders, usuallylyophilized, including excipients which on dissolution will provide areagent solution having the appropriate concentration.

Also provided are diagnostic or detection reagents and kits comprisingone or more such reagents for use in a variety of detection assays,including for example, immunoassays such as ELISA (sandwich-type orcompetitive format). The kit's components may be pre-attached to a solidsupport, or may be applied to the surface of a solid support when thekit is used. In some embodiments of the invention, the signal generatingmeans may come pre-associated with an antibody or fragment of theinvention or may require combination with one or more components, e.g.,buffers, antibody-enzyme conjugates, enzyme substrates, or the like,prior to use. Kits may also include additional reagents, e.g., blockingreagents for reducing nonspecific binding to the solid phase surface,washing reagents, enzyme substrates, and the like. The solid phasesurface may be in the form of a tube, a bead, a microtiter plate, amicrosphere, or other materials suitable for immobilizing proteins,peptides, or polypeptides. In particular aspects, an enzyme thatcatalyzes the formation of a chemilluminescent or chromogenic product orthe reduction of a chemilluminescent or chromogenic substrate is acomponent of the signal generating means. Such enzymes are well known inthe art. Kits may comprise any of the capture agents and detectionreagents described herein. Optionally the kit may also compriseinstructions for carrying out the methods of the invention.

Also provided is a kit comprising an anti-TIGIT antibody (e.g.,humanized antibody) or antigen-binding fragment thereof packaged in acontainer, such as a vial or bottle, and further comprising a labelattached to or packaged with the container, the label describing thecontents of the container and providing indications and/or instructionsregarding use of the contents of the container to treat one or moredisease states as described herein.

In one aspect, the kit is for treating cancer and comprises ananti-TIGIT antibody (e.g., humanized antibody) or antigen-bindingfragment thereof and a further therapeutic agent or a vaccine. The kitmay optionally further include a syringe for parenteral, e.g.,intravenous, administration. In another aspect, the kit comprises ananti-TIGIT antibody (e.g., humanized antibody) or antigen-bindingfragment thereof and a label attached to or packaged with the containerdescribing use of the antibody or fragment with the vaccine or furthertherapeutic agent. In yet another aspect, the kit comprises the vaccineor further therapeutic agent and a label attached to or packaged withthe container describing use of the vaccine or further therapeutic agentwith the anti-TIGIT antibody or fragment. In certain embodiments, ananti-TIGIT antibody and vaccine or further therapeutic agent are inseparate vials or are combined together in the same pharmaceuticalcomposition.

As discussed above in the combination therapy section, concurrentadministration of two therapeutic agents does not require that theagents be administered at the same time or by the same route, as long asthere is an overlap in the time period during which the agents areexerting their therapeutic effect. Simultaneous or sequentialadministration is contemplated, as is administration on different daysor weeks.

The therapeutic and detection kits disclosed herein may also be preparedthat comprise at least one of the antibody, peptide, antigen-bindingfragment, or polynucleotide disclosed herein and instructions for usingthe composition as a detection reagent or therapeutic agent. Containersfor use in such kits may typically comprise at least one vial, testtube, flask, bottle, syringe or other suitable container, into which oneor more of the detection and/or therapeutic composition(s) may beplaced, and preferably suitably aliquoted. Where a second therapeuticagent is also provided, the kit may also contain a second distinctcontainer into which this second detection and/or therapeuticcomposition may be placed. Alternatively, a plurality of compounds maybe prepared in a single pharmaceutical composition, and may be packagedin a single container means, such as a vial, flask, syringe, bottle, orother suitable single container. The kits disclosed herein will alsotypically include a means for containing the vial(s) in closeconfinement for commercial sale, such as, e.g., injection or blow-moldedplastic containers into which the desired vial(s) are retained. Where aradiolabel, chromogenic, fluorigenic, or other type of detectable labelor detecting means is included within the kit, the labeling agent may beprovided either in the same container as the detection or therapeuticcomposition itself, or may alternatively be placed in a second distinctcontainer means into which this second composition may be placed andsuitably aliquoted. Alternatively, the detection reagent and the labelmay be prepared in a single container means, and in most cases, the kitwill also typically include a means for containing the vial(s) in closeconfinement for commercial sale and/or convenient packaging anddelivery.

A device or apparatus for carrying out the detection or monitoringmethods described herein is also provided. Such an apparatus may includea chamber or tube into which sample can be input, a fluid handlingsystem optionally including valves or pumps to direct flow of the samplethrough the device, optionally filters to separate plasma or serum fromblood, mixing chambers for the addition of capture agents or detectionreagents, and optionally a detection device for detecting the amount ofdetectable label bound to the capture agent immunocomplex. The flow ofsample may be passive (e.g., by capillary, hydrostatic, or other forcesthat do not require further manipulation of the device once sample isapplied) or active (e.g., by application of force generated viamechanical pumps, electroosmotic pumps, centrifugal force, or increasedair pressure), or by a combination of active and passive forces.

In further embodiments, also provided is a processor, a computerreadable memory, and a routine stored on the computer readable memoryand adapted to be executed on the processor to perform any of themethods described herein. Examples of suitable computing systems,environments, and/or configurations include personal computers, servercomputers, hand-held or laptop devices, multiprocessor systems,microprocessor-based systems, set top boxes, programmable consumerelectronics, network PCs, minicomputers, mainframe computers,distributed computing environments that include any of the above systemsor devices, or any other systems known in the art.

GENERAL METHODS

Standard methods in molecular biology are described Sambrook, Fritschand Maniatis (1982 & 1989 2^(nd) Edition, 2001 3^(rd) Edition) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning,3^(rd) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego,Calif.). Standard methods also appear in Ausbel, et al. (2001) CurrentProtocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. NewYork, N.Y., which describes cloning in bacterial cells and DNAmutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2),glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed (Coligan, et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,chemical modification, post-translational modification, production offusion proteins, glycosylation of proteins are described (see, e.g.,Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2,John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) CurrentProtocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY,N.Y., pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for LifeScience Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech(2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production,purification, and fragmentation of polyclonal and monoclonal antibodiesare described (Coligan, et al. (2001) Current Protcols in Immunology,Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999)Using Antibodies, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Harlow and Lane, supra). Standard techniques forcharacterizing ligand/receptor interactions are available (see, e.g.,Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, JohnWiley, Inc., New York).

Monoclonal, polyclonal, and humanized antibodies can be prepared (see,e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ.Press, New York, N.Y.; Kontermann and Dubel (eds.) (2001) AntibodyEngineering, Springer-Verlag, New York; Harlow and Lane (1988)Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp. 139-243; Carpenter, et al. (2000) J.Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang et al.(1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem.272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote andWinter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

An alternative to humanization is to use human antibody librariesdisplayed on phage or human antibody libraries in transgenic mice(Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995)Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377;Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Kay et al. (1996)Phage Display of Peptides and Proteins: A Laboratory Manual, AcademicPress, San Diego, Calif.; de Bruin et al. (1999) Nature Biotechnol.17:397-399).

Single chain antibodies and diabodies are described (see, e.g., Maleckiet al. (2002) Proc. Natl. Acad. Sci. USA 99:213-218; Conrath et al.(2001) J. Biol. Chem. 276:7346-7350; Desmyter et al. (2001) J. Biol.Chem. 276:26285-26290; Hudson and Kortt (1999) J. Immunol. Methods231:177-189; and U.S. Pat. No. 4,946,778). Bifunctional antibodies areprovided (see, e.g., Mack, et al. (1995) Proc. Natl. Acad. Sci. USA92:7021-7025; Carter (2001) J. Immunol. Methods 248:7-15; Volkel, et al.(2001) Protein Engineering 14:815-823; Segal, et al. (2001) J. Immunol.Methods 248:1-6; Brennan, et al. (1985) Science 229:81-83; Raso, et al.(1997) J. Biol. Chem. 272:27623; Morrison (1985) Science 229:1202-1207;Traunecker, et al. (1991) EMBO J. 10:3655-3659; and U.S. Pat. Nos.5,932,448, 5,532,210, and 6,129,914).

Bispecific antibodies are also provided (see, e.g., Azzoni et al. (1998)J. Immunol. 161:3493; Kita et al. (1999) J. Immunol. 162:6901; Merchantet al. (2000) J. Biol. Chem. 74:9115; Pandey et al. (2000) J. Biol.Chem. 275:38633; Zheng et al. (2001) J. Biol Chem. 276:12999; Propst etal. (2000) J. Immunol. 165:2214; Long (1999) Ann. Rev. Immunol. 17:875).

Purification of antigen is not necessary for the generation ofantibodies. Animals can be immunized with cells bearing the antigen ofinterest. Splenocytes can then be isolated from the immunized animals,and the splenocytes can fused with a myeloma cell line to produce ahybridoma (see, e.g., Meyaard et al. (1997) Immunity 7:283-290; Wrightet al. (2000) Immunity 13:233-242; Preston et al., supra; Kaithamana etal. (1999) J. Immunol. 163:5157-5164).

Antibodies can be conjugated, e.g., to small drug molecules, enzymes,liposomes, polyethylene glycol (PEG). Antibodies are useful fortherapeutic, diagnostic, kit or other purposes, and include antibodiescoupled, e.g., to dyes, radioisotopes, enzymes, or metals, e.g.,colloidal gold (see, e.g., Le Doussal et al. (1991) J. Immunol.146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsingand Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J.Immunol. 168:883-889).

Methods for flow cytometry, including fluorescence activated cellsorting (FACS), are available (see, e.g., Owens, et al. (1994) FlowCytometry Principles for Clinical Laboratory Practice, John Wiley andSons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2^(nd) ed.;Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, JohnWiley and Sons, Hoboken, N.J.). Fluorescent reagents suitable formodifying nucleic acids, including nucleic acid primers and probes,polypeptides, and antibodies, for use, e.g., as diagnostic reagents, areavailable (Molecular Probes (2003) Catalogue, Molecular Probes, Inc.,Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Standard methods of histology of the immune system are described (see,e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology andPathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) ColorAtlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.;Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, NewYork, N.Y.).

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available (see, e.g.,GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG WisconsinPackage (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp.,Crystal Bay, Nev.); Menne, et al. (2000) Bioinformatics 16: 741-742;Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren,et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.14:4683-4690).

Example 1 Generation of Rat and Mouse Anti-hTIGIT Antibodies

To generate antibodies to human TIGIT, Lewis rats were immunized withhis-tagged human TIGIT recombinant protein from Sino BiologicalsCat#10917-H08H) using RIBI adjuvant and footpad injection on a biweeklyschedule. Alternatively, Balb/C mice were immunized with human Fc-taggedhuman TIGIT recombinant protein using RIBI adjuvant and footpadinjection on a biweekly schedule. Immunized animals were bled and serumtiters determined for binding to human TIGIT transfected CHOK1 cellsusing a cell-based ELISA (described below). The animals with the highesttiters were given a final boost with recombinant protein and drainingpopliteal lymph nodes isolated four days later. Hybridomas weregenerated by electrofusion of isolated lymphocytes with the myelomafusion partner P3×63-AG8.653 using the Cytopulse Hybrimmuneelectrofusion system. Fused cells were plated in 96-well plates inDMEM/F12, 15% BCS, HAT, IL-6, OPI supplement, and gentamycin.

Hybridoma supernatants were assayed for binding to human TIGITexpressing CHOK1 cells and cross-reactivity to rhesus TIGIT expressingCHO cells using a cell-based ELISA format. Human TIGIT and rhesus TIGITexpressing CHO-K1 cells were plated in 96-well tissue-culture plates in50 μl of DMEM/F12, 10% BCS and gentamycin (CHO-K1 media). Cells wereplated at either 2×10⁴ cells/well two days prior to the assay or 4×10⁴cells/well one day prior to the assay. Media was removed from the wellsprior to the assay and 50 μl of hybridoma supernatant added. Hybridomasupernatants were incubated for 30-60 minutes at room temperature andwashed 3 times with PBS/0.05% Tween 20 using a cell ELISA washingprotocol on the Biotek EL405× Select CW plate washer. Fifty microlitersof the detection antibody (HRP-conjugated goat anti-rat IgG (SouthernBiotech cat#3030-05) or HRP-conjugated goat anti-mouse IgG (SouthernBiotech cat#1043-05)), was added at a 1:2000 dilution in CHO-K1 mediaand incubated at room temperature for 30-60 minutes. Assay plates werewashed as above and developed with TMB and stopped with TMB stopsolution (KPL cat#50-85-06) or 0.1N phosphoric acid. The absorbance at450 nm-620 nm was determined. Positive clones were reactive to bothhuman TIGIT and rhesus TIGIT transfected CHO-K1 cells, and were negativefor binding to parental CHO-K1 cells. In these assays, if an antibodyshowed binding to parental (untransfected) CHO-K1 cells; we discardedthat antibody in screening as not specific to TIGIT.

Positive hybridomas were subcloned by limiting dilution or subcloned byplating hybridomas in semi-solid media and clones picked on theClonePix® (Genetix). Two rounds of subcloning were performed on theparental hybridomas. Final subclones were grown in small-scale culturesin serum-free hybridoma production medium and purified to generatepurified antibody for further characterization.

Using these methods, about 819 hybridomas were generated.

Example 2 Characterization of Anti-hTIGIT Antibodies

Supernatants from positive clones were tested for their ability to blockrecombinant human CD155-huFc protein binding to hTIGIT CHOK1 cells in acell-based ELISA format. Human TIGIT-CHO-K1 cells were plated in 96-wellplates as described above. Media was removed from the plates and 50 μlof hybridoma supernatant was incubated with the human TIGIT CHO-K1 cellsat 4° C. for 30 minutes. Fifty microliters of human CD155-huFc was addedto the plate for a final concentration of 0.5 μg/ml of human CD155-huFcand incubated for 30 minutes at 4° C. Assay plates were washed 3 timeswith PBS/0.05% Tween-20 as above. Binding of human CD155-huFc to thehTIGIT-CHOK1 cells was detected using an HRP-conjugate F(ab)′2 goatanti-human IgG secondary antibody (Jackson 109-036-098) at 1:2000dilution in CHO-K1 media. Plates were developed using TMB and stoppedusing TMB Stop Solution as described above and the A450-620 nmdetermined.

A rat antibody generated according to the above described method isreferred to as 14A6, and was derived from clone LB155.14A6.G2.A8. Thisrat antibody (14A6) is of the IgG2/kappa isotype and comprises the heavychain variable region of SEQ ID NO:7 and the light chain variable regionof SEQ ID NO:8. Purified 14A6 antibody binds to human TIGIT and rhesusTIGIT as determined by cell-based ELISA binding to human TIGIT andrhesus TIGIT-CHOK1 cells (FIG. 1) using the methods described above. (Anisotype control antibody did not show any binding (data not shown.)Purified 14A6 antibody can also block the hTIGIT and hCD155 interactionusing a cell-based ELISA blocking assay (FIG. 3) using the methoddescribed above.

A mouse antibody generated according to the above described method isreferred to as 28H5, and was derived from clone TC167.28H5.H5. Thismouse antibody (28H5) is of the IgG1/kappa isotype and comprises theheavy chain variable region of SEQ ID NO:63 and the light chain variableregion of SEQ ID NO:64. Purified 28H5 antibody also binds to human TIGITand rhesus TIGIT as determined by cell-based ELISA binding to humanTIGIT and rhesus TIGIT-CHOK1 cells (FIG. 2) using the methods describedabove. Purified 28H5 antibody also block the hTIGIT and hCD155interaction using a cell-based ELISA blocking assay (FIG. 3) using themethod described above.

Another mouse antibody generated according to the above described methodis referred to as 31C6, and was derived from clone MEB125.31C6.A1.205.This mouse antibody (31C6) is of the IgG1/kappa isotype and comprisesthe heavy chain variable region of SEQ ID NO:94 and the light chainvariable region of SEQ ID NO:95. Purified 31C6 antibody also binds tohuman TIGIT and rhesus TIGIT as determined by cell-based ELISA bindingto human TIGIT and rhesus TIGIT-CHOK1 cells (FIG. 4) using the methodsdescribed above. Purified 31C6 antibody also block the hTIGIT and hCD155interaction using a cell-based ELISA blocking assay (FIG. 4) using themethod described above.

Affinity Determination for Binding of Parental (Non-Human) Anti-TIGITAntibodies to Human TIGIT Recombinant Protein:

The kinetic binding activity of anti-human TIGIT antibodies 14A6 and31C6 (made as described in Example 1) and of commercial antibody MBSA43was measured by surface plasmon resonance using a Biacore T200 system(Biacore, GE Healthcare, Piscataway, N.J.). Approximately 5000 RU ofAnti-mouse IgG, GE Healthcare Catalog Number BR-1008-38, orapproximately 13,000 RU of Goat Anti-Rat IgG Fc gamma, FragmentSpecific, Jackson ImmunoResearch Catalog Number 112-006-071, wasimmobilized via amine coupling chemistry onto a Series S CM5 sensorchip, catalog number BR-1005-30. Mouse anti-human TIGIT clones, 31C6 andMBSA43, were each injected over the immobilized anti-mouse surfaces at 1ug/mL for a capture level of 40 RU. Rat anti-human TIGIT clone 14A6 wasinjected over the immobilized anti-rat surfaces at 1 ug/mL for a capturelevel of 40 RU. HBS-EP+ buffer (BR-1006-69) was used as the runningbuffer with a flow rate of 30 μL/min. Varying concentrations of humanTIGIT-His protein or TIGIT-Fc protein, ranging from 0.29 nM to 40 nM, ata flow rate of 45 μL/min were injected over the antibody surfaces.Following each Mouse anti-human TIGIT injection cycle for clones 31C6and MBSA43, the Series S CM5 chip surface was regenerated using onethree-minute injection of 10 mM Glycine pH 1.7 at a flow rate of 10μL/min. Following each Rat anti-human TIGIT injection cycle, the SeriesS CM5 chip surface was regenerated using one 20-second injection of 10mM Glycine pH 1.5 followed by two 10-second injections of 12.5 mM NaOHat a flow rate of 60 μL/min.

Background subtraction binding sensorgrams were used for analyzing therate constant of association (k_(a)) and dissociation (k_(d)), and theequilibrium dissociation constant K_(D). The resulting data sets werefitted with a 1:1 Langmuir Binding Model using the Biacore T200evaluation software (version 2.0). Table 3 summarizes the affinities forthe anti-human TIGIT antibodies to human TIGIT-His protein and TIGIT-Fcprotein.

TABLE 3 Measurement of Affinity for anti-Human TIGIT Antibodies to humanTIGIT-His protein and TIGIT-Fc protein using BIAcore. BIAcore KD BIAcoreKD (human TIGIT-his) (human TIGIT-Fc) Clone (pM) (pM) 14A6 (rIgG2a/K)3.09 3.12 31C6 (mIgG1/K) 34.4 10.9 Comparator MBSA43 36.3 16.5 (mIgG1)

Example 3 In Vitro T-Cell Activity Assay for Antagonistic Anti-hTIGITAntibodies

Of the antibodies tested, approximately 352 monoclonal antibodies shownto block binding of CD155-Fc to CHO cells expressing hTIGIT werescreened for their capacity enhance T cell activity in vitro usingcell-based functional assays.

One assay we developed to characterize the functional consequence ofblocking human TIGIT receptor utilized Jurkat cells, an immortalizedline of human T lymphocyte cells (clone, E6-1; ATCC TIB-152), engineeredto over-express human TIGIT (hTIGIT-Jurkat) which were co-cultured withTHP-1 cells, a human monocytic cell line in the presence or absence ofone of the TIGIT ligands, CD155 and CD112. hTIGIT-Jurkat cellsco-cultured with THP-1 cells and stimulated with plate-bound anti-CD3mAb produce IL-2, but when TIGIT ligand (CD155 or CD112) is added to theco-culture, IL-2 levels were reduced in a ligand-dependent manner.Treatment with antibodies that blocked the CD155- or CD112-TIGITinteraction, such as a commercially available anti-hTIGIT Ab, cloneMBSA43 (eBioscience Cat#12-9500-42), rescues IL-2 production in thisassay in a dose-dependent fashion (FIG. 5).

96-well flat-bottom plates were coated with mouse anti-human CD3antibody (1 ug/ml in PBS; Clone HIT3a; BD Pharmingen Cat#555336)overnight at 4° C. The next day, hTIGIT-Jurkat cells (50,000) wereplated in the pre-coated plates and pre-incubated for 30-60 minutes withmAb at varying concentrations. THP-1 cells (50,000) were added to theculture followed by either CD155-Fc (ECD of human CD155 fused to humanFc; 1.0 μg/ml) or CD112-Fc (ECD of human CD112 fused to human Fc; 0.5μg/ml). After incubation for 18-24 h at 37° C. and 5.0% CO₂, IL-2 levelswere assessed in culture supernatants by Meso Scale (Human IL-2 TissueCulture MESO Kit: Cat#K151AHB-2). Treatment with MBSA43 (10 μg/ml)rescues IL-2 to a level equal to when activated hTIGIT Jurkat cells arecultured with THP-1 in the absence of CD155 or CD112.

As shown in FIG. 5, a titration of the anti-hTIGIT clone 14A6 from 30μg/ml down to 0.04 μg/ml gave an EC50 of 0.190 μg/ml as compared toMBSA43 at 0.24 μg/ml using this assay. Clone 14A6 (30 μg/ml) rescuedIL-2 to 82% of MBSA43 (10 μg/ml).

Also shown in FIG. 5, a titration of the anti-hTIGIT clone 28H5 from 30μg/ml down to 0.04 μg/ml gave an EC50 of 0.24 μg/ml as compared toMBSA43 at 0.24 μg/ml using this assay.

Also shown in FIG. 6, a titration of the anti-hTIGIT clone 31C6 from 30μg/ml down to 0.04 μg/ml gave an EC50 of 0.14 μg/ml as compared toMBSA43 at 0.24 μg/ml using this assay. Clone 31C6 (30 μg/ml) rescuedIL-2 to 118% of MBSA43 (10 μg/ml).

Of the 352 monoclonal antibodies tested, approximately 113 were able toenhance T cell activity in vitro using cell-based functional assays.

Example 4 In Vitro T-Cell Activity Assay for Antagonistic Anti-hTIGITAntibodies

We further analyzed the relative functional antagonism of TIGIT andrescue of T cell activation and effector function using a primary humanT cell line expressing endogenous TIGIT upon T cell receptor (TCR)activation. The BC4-clone 49 human T cell line is an allo-antigenspecific human CD4+ T cell clone expressing a TCR specific for HLA-classII MHC molecules expressed on the EBV transformed cell line, JY(HLA-DR1,4). The BC4-clone 49 human T cell line requires re-stimulationwith allo antigen every two weeks, using irradiated (5000 Rads) JYstimulator cells, irradiated PBMC (4000 Rads) isolated from two buffycoats and PHA-P (2.5 μg/ml; Sigma), as well as 10 ng/ml human IL-2 (R&D)supplement every 3-4 day following restimulation and expansion. NumerousT cell clones (BC1-6, BC4-27, BC4-49) were analyzed by PCR and then byflow cytometry for relative expression of TIGIT following restimulationand BC4-clone 49 was selected as it had the highest expression of TIGITcompared to other clones. The relative expression and kinetics of TIGIT,CD226, CD96 and CD155 were monitored following re-stimulation withirradiated (5000 Rads) JY stimulator cells, irradiated PBMC and PHA-P toassess the best time to assay antagonist mAbs for relativeTIGIT-antagonist activity. Peak expression of TIGIT was observed at 3-4days post restimulation, while expression of CD226 and CD96 decreasedover the same time period. Expression of TIGIT then decreased, whileexpression of CD226 and CD96 increased again by day 6 post-restimulation(FIG. 7). Accordingly, candidate TIGIT antagonist mAbs were assessed at3-4 days post-restimulation. Transfectant JY overexpressing human CD155were generated using pMX->huTIGIT retroviral vectors as a means tosuppress BC4-clone 47 T cell responses in the primary T cell bioassayand to assess the relative capacity of anti-TIGIT mAbs to antagonizeCD155 activation of TIGIT and rescue T cell proliferation and IFNγ threedays later.

The primary T cell assay for relative anti-hTIGIT antagonist mAbs wasset up as follows. Human CD4⁺ T cell line BC4-clone 49 was co-culturedwith CD155-Fc blocking mAbs specific for hTIGIT at variousconcentrations (33, 11, 3.7, 1.2, 0.4 and 0.1 μg/ml) for 30 minutes andthen plated in round-bottom 96-well plates (2×10⁴ c/well). JY-hCD155transfectants, expressing the allo-antigen and high levels of hCD155,were irradiated (5000 Rads), washed and then added to the wellscontaining the anti-TIGIT mAb treated BC4-clone 49 T cells for a finalconcentration of 1×10⁴ (2:1 T cell: target cell ratio) or 5×10³ (1:4 Tcell: target cell ratio) in a total volume of 200 μl/well. Controlsincluded no mAb treatment, co-culture with isotype mAb treated T cells,T cell only and JY-CD155 cells only. After three or four days ofco-culture, supernatants were harvested for interferon-gamma (IFNγ)cytokine quantification, and the T cells were pulsed 6 hours withtritiated-thymidine to assess relative proliferation.

As shown in FIGS. 8A and 8B, anti-hTIGIT mAb 14A6, 28H5 and 31C6treatment of BC4-clone 49 T cells resulted in rescue of IFNγ andproliferative responses as assessed by increased responses compared toisotype and untreated T cells. Commercial antibody MBSA43 and antibodies37D10 and 25G10 (anti-TIGIT antibodies produced as described inExample 1) also show activity in this assay. The increase in IFNγproduction after treatment with 14A6, 28H5 and 31C6 was on about 2 foldon average.

Example 5 Anti-Tumor Activity of Anti-TIGIT and Anti-PD1 Antibodies inAnimal Tumor Model

Mice:

Approximately seven to eight week old female BALB/cAnN mice with anaverage body weight of 20 grams were obtained from Taconic Laboratory(Germantown, N.Y.). Conventional animal chow and water were provided adlibitum.

Antibody Reagents:

A monoclonal antibody of murine isotype IgG1 against murine PD-1 andisotype controls were obtained from internal sources as frozen (−80° C.)stocks. The rat anti-mouse TIGIT (GIGD7) antibody was obtained fromeBioscience as a 4° C. stock. The IgG1 isotype control was a mousemonoclonal antibody specific for adenoviral hexon 25. The IgG2a isotypecontrol was a rat monoclonal antibody specific for beta-galactosidase.Murine isotype IgG1 is the murine counterpart isotype to human isotypeIgG4.

Formulations of Antibody Reagents:

The formulation buffers were specific for each antibody to stabilizeproteins and prevent precipitation. The formulations were as follows:mIgG1:75 mM NaCl, 10 mM sodium phosphate, 3% sucrose, pH7.3;anti-PD-1:20 mM sodium acetate, 7% sucrose, pH5.5; rat IgG2a: 20 mMsodium acetate, 9% sucrose pH 5.5; and anti-TIGIT (GIGD7): PBS pH 7.0.

Tumor Cell Line Preparation and Implant:

CT26 colon carcinoma cells were cultured in RPMI medium supplementedwith 10% heat-inactivated fetal bovine serum. 3×10⁵ log-phase andsub-confluent CT26 cells were injected subcutaneously (SC) in a 100 μLvolume of serum-free RPMI in the right lower dorsal flank of each mouse.Mice were first shaved with electronic clippers in the area that wouldbe used for the implant.

CT-26 is a murine colorectal adenocarcinoma cell line syngeneic to theBALB/c mouse strain. CT-26 is a relevant model system for evaluating themechanism of action for an anti-PD-1 antibody because of thetranslatable molecular profile of this tumor post-anti-PD-1 therapy.

Tumor Measurements and Body Weights:

Tumors were measured the day before the first dose and twice a weekthereafter. Tumor length and width were measured using electroniccalipers and tumor volume determined using the formula Volume(mm³)=0.5×Length×Width² where length is the longer dimension. Mice wereweighed periodically to monitor general health but also to estimateactual mg/kg dose delivery per mouse where needed. Before treatment,mice were weighed and tumors from individual mice were measured. Toprevent bias, any outliers by weight or tumor volume were removed andthe remaining mice randomized into various treatment groups withequivalent mean tumor size. When the mean tumor volume in the CT26tumor-bearing mice reached ˜100 mm³, around 7 days post implant, animalswere randomized into treatment groups of 10 mice per group and dosingwas started. Animals were administered at the dosing concentrationsspecified below.

Dosing Solution Preparation, Administration, and Analyses:

Frozen stocks of the antibodies to be tested in the animal model werethawed and transferred to wet ice. To avoid repeated freeze thaw, eachvial of stock was thawed once and aliquots made in volumes sufficientfor one time use. Polypropylene, low adhesion tubes were used for thispurpose. The aliquots were snap frozen in dry ice and stored at −80° C.Before each dosing, one aliquot was thawed and diluted to nominalconcentration in the appropriate diluent and dosed immediately. Aliquotsof dosing solutions were snap frozen in dry ice and stored at −80° C.until analyses. Dosing solutions were assessed using the Meso ScaleDiscovery (MSD®, Rockville, Md.) platform which is based on multi-arraytechnology; a combination of electrochemiluminescence detection andpatterned arrays.

Dosing Anti-TIGIT/Anti-PD1 Treatment Results:

CT26 tumor-bearing BALB/cAnN mice were administered rat anti-mouse TIGIT(GIGD7) at a 20 mg/kg dose, IP, every 4 days for each of 5 cycles. Ananti-mouse PD-1 (described above) was administered at a 10 mg/kg dose,IP, every 4 days for each of 5 cycles. Post dosing, animals continued tobe monitored and tumor volumes were measured out to 36 days. Treatmentwas started once the tumor size averaged 100 mm³ (75 mm³-115 mm³).Tumors were measured twice weekly. As demonstrated by the results, whichare shown in FIG. 9, the mean anti-tumor response of combination therapywith the PD-1 antagonist and TIGIT antagonist was greater than theanti-tumor response observed with either anti-PD1 single agent (p<0.05)or anti-TIGIT single agent (p<0.005) treatment. For single agentanti-TIGIT treatment, 21% tumor growth inhibition (TGI) was observed atday 14. For single agent anti-PD1 treatment, 52% TGI was observed at day14. Combination treatment resulted in 85% TGI at day 14 and demonstrated40% complete regressions (CR) such that no measurable tumor remained in4 out of 10 mice by day 36. The anti-tumor efficacy with either antibodydelivered as monotherapy was 0-10% CR.

Example 6 Humanization of Antibodies

The rat 14A6 and the mouse 31C6 antibody were humanized using methodsdescribed in the specification. From the rat antibody 14A6, thefollowing humanized variable heavy chains were constructed: SEQ ID NOs:9-24 and SEQ ID NOs: 37-47; and the following humanized variable lightchains were constructed: SEQ ID NOs: 25-30 and SEQ ID NOs: 48-52. Frommouse antibody 31C6, the following humanized variable heavy chains wereconstructed: SEQ ID NOs: 124-129; and the following humanized variablelight chains were constructed: SEQ ID Nos: 130-133.

Example 7 Effect of Fc Isotype on the Anti-Tumor Activity of Anti-TIGITAntibodies in Animal Tumor Model

Mice:

Approximately seven to eight week old female BALB/cAnN mice with anaverage body weight of 20 grams were obtained from Taconic Laboratory(Germantown, N.Y.). Conventional animal chow and water were provided adlibitum.

Antibody Reagents:

-   -   Murine anti-mouse PD1 antibody—IgG1 subtype    -   Rat anti-mouse TIGIT antibody (18G10)-rat IgG1 subtype. This        antibody is described in the Figures as 18G10 parental. The        18G10 antibody has the VH sequence of SEQ ID NO:136 and the VL        sequence of SEQ ID NO:137.    -   Chimeric rat anti-mouse TIGIT antibody (18G10)-comprising a        mouse Fc region of mIgG1 subtype. This antibody is described in        the Figures as 18G10-G2a. (Murine isotype IgG1 is the murine        counterpart isotype to human isotype IgG4.)    -   Chimeric rat anti-mouse 18G10 TIGIT antibody—comprising a mouse        Fc region of mIgG2a subtype. This antibody is described in the        Figures as 18G10-G2a. (Murine isotype IgG2a is the murine        counterpart isotype to human isotype IgG1.)    -   Isotype control murine IgG1 antibody (mouse IgG1 isotype-matched        control monoclonal antibody specific for adenoviral hexon 25))    -   Isotype control rat IgG1 antibody (rat IgG1 isotype-matched        control monoclonocal antibody specific for human IL-4)    -   Isotype control murine IgG2a antibody (mouse IgG2a        isotype-matched control monoclonal antibody specific for        adenoviral hexon 25).

Formulations of Antibody Reagents:

The formulations were as follows:

-   -   mIgG1:75 mM NaCl, 10 mM sodium phosphate, 3% sucrose, pH7.4;    -   anti-PD-1:20 mM sodium acetate, 9% sucrose, pH5.5; mIgG2a: 75 mM        NaCl, 10 mM sodium phosphate, 3% sucrose, pH7.3;    -   rat IgG1:20 mM sodium acetate, 7% sucrose pH 5.5; 18G10:20 mM        NaAc, 100 mM NaCl, 3% sucrose; 18G10-G1:20 mM NaAc, 9% sucrose,        pH 5.5; 18G10-G2a: 20 mM NaAc, 9% sucrose, pH 5.5.

Tumor Cell Line Preparation and Implant:

CT26 colon carcinoma cells were cultured in RPMI medium supplementedwith 10% heat-inactivated fetal bovine serum. 3×10⁵ log-phase andsub-confluent CT26 cells were injected subcutaneously (SC) in a 100 μLvolume of serum-free RPMI in the right lower dorsal flank of each mouse.Mice were first shaved with electronic clippers in the area that wouldbe used for the implant.

CT-26 is a murine colorectal adenocarcinoma cell line syngeneic to theBALB/c mouse strain. CT-26 is a relevant model system for evaluating themechanism of action for an anti-PD-1 antibody because of thetranslatable molecular profile of this tumor post-anti-PD-1 therapy.

Tumor Measurements and Body Weights:

Tumors were measured the day before the first dose and twice a weekthereafter. Tumor length and width were measured using electroniccalipers and tumor volume determined using the formula Volume(mm³)=0.5×Length×Width² where length is the longer dimension. Mice wereweighed periodically to monitor general health but also to estimateactual mg/kg dose delivery per mouse where needed. Before treatment,mice were weighed and tumors from individual mice were measured. Toprevent bias, any outliers by weight or tumor volume were removed andthe remaining mice randomized into various treatment groups withequivalent mean tumor size.

Dosing Solution Preparation, Administration, and Analyses:

Frozen stocks of the antibodies to be tested in the animal model werethawed and transferred to wet ice. To avoid repeated freeze thaw, eachvial of stock was thawed once and aliquots made in volumes sufficientfor one time use. Polypropylene, low adhesion tubes were used for thispurpose. The aliquots were snap frozen in dry ice and stored at −80° C.Before each dosing, one aliquot was thawed and diluted to nominalconcentration in the appropriate diluent and dosed immediately. Aliquotsof dosing solutions were snap frozen in dry ice and stored at −80° C.until analyses. Dosing solutions were assessed using the Meso ScaleDiscovery (MSD®, Rockville, Md.) platform which is based on multi-arraytechnology; a combination of electrochemiluminescence detection andpatterned arrays.

Dosing Anti-TIGIT/Anti-PD1 Treatment Results:

CT26 tumor-bearing BALB/cAnN mice were randomized into 10 treatmentgroups when the mean tumor volume of these mice reached a tumor sizeaverage of 100 mm3 (80 mm3-119 mm3): (1) muIgG1 isotype control+rat IgG1isotype control; (2) muIgG1 isotype control+ muIgG2a isotype control;(3) muDX400+ rat IgG1 isotype control; (4) muDX400+ muIgG2a isotypecontrol; (5) muIgG1 isotype control+18G10; (6) muIgG1 isotypecontrol+18G10-G1; (7) muIgG1 isotype control+18G10-G2a; (8)muDX400+18G10; (9) muDX400+18G10_G1; (10) muDX400+18G10_G2a. Animalswere administered rat anti-mouse TIGIT (18G10) or chimeric anti-TIGITantibodies 18G10-G1 or 18G10-G2a (described above) at a 18 mg/kg dose,IP, every 4 days for each of 6 cycles. An anti-mouse PD-1 (describedabove) was administered at a 10 mg/kg dose, IP, every 4 days for each of6 cycles. Post dosing, animals continued to be monitored and tumorvolumes were measured out to 76 days for some treatment groups. Tumorswere measured twice weekly. The results are shown in FIGS. 10 and 11.Single agent anti-TIGIT treatment using 18G10-G2a showed 44% tumorgrowth inhibition (TGI) at day 13; while 18G10 parental antibody showed38% and the 18G10-G1 antibody showed 36%. For single agent anti-PD1treatment combined with isotype control rIgG1, 51% TGI was observed atday 13. When anti-PD-1 was combined with isotype control muIgG2a, 50%TGI and 10% complete regressions (CR) were observed at day 18, such that1 complete response out of 10 animals was observed. The combination ofanti-PD1 and parental 18G10 showed 80% TGI at day 13 and demonstrated300% CR by day 39. The combination of anti-PD1 and 18G10-G1 showed 59%TGI at day 13 and demonstrated 10% CR by day 13. The combination ofanti-PD1 and 18G10-G2a showed 88% TGI at day 13 and demonstrated 60% CRat day 63. The combination of anti-PD1 and 18G10-G2a showed greateranti-tumor activity and more complete regressions compared tocombinations of anti-PD1 with 18G10 parental or 18G10-G1. There was nosignificant body weight loss associated with administration of singleagents or combination therapy indicating that treatments were welltolerated.

Example 8 Effect of Fc Isotype on the Anti-Tumor Activity of Anti-TIGITAntibodies in Animal Tumor Model

The Experiment described in Example 7 was repeated, except that the rat18G10 antibody was substituted with rat antibody 11A11.

Mice:

Approximately seven to eight week old female BALB/cAnN mice with anaverage body weight of 20 grams were obtained from Taconic Laboratory(Germantown, N.Y.). Conventional animal chow and water were provided adlibitum.

Antibody Reagents:

-   -   Murine anti-mouse PD1 antibody—IgG1 subtype    -   Rat anti-mouse TIGIT antibody (11A11)-rat IgG1 subtype. This        antibody is described in the Figures as 11A11 parental. The        11A11 antibody has the VH sequence of SEQ ID NO:138 and the VL        sequence of SEQ ID NO:139.    -   Chimeric rat anti-mouse TIGIT antibody (11A11)-comprising a        mouse Fc region of mIgG1 subtype. This antibody is described in        the Figures as 11A11-G2a. (Murine isotype IgG1 is the murine        counterpart isotype to human isotype IgG4.)    -   Chimeric rat anti-mouse 11A11 TIGIT antibody—comprising a mouse        Fc region of mIgG2a subtype. This antibody is described in the        Figures as 11A11-G2a. (Murine isotype IgG2a is the murine        counterpart isotype to human isotype IgG1.)    -   Isotype control murine IgG1 antibody (mouse IgG1 isotype-matched        control monoclonal antibody specific for adenoviral hexon 25))    -   Isotype control rat IgG1 antibody (rat IgG1 isotype-matched        control monoclonocal antibody specific for human IL-4)    -   Isotype control murine IgG2a antibody (mouse IgG2a        isotype-matched control monoclonal antibody specific for        adenoviral hexon 25).

Formulations of Antibody Reagents:

The formulations were as follows:

-   -   mIgG1:75 mM NaCl, 10 mM sodium phosphate, 3% sucrose, pH7.4;    -   anti-PD-1:20 mM sodium acetate, 9% sucrose, pH5.5; mIgG2a: 75 mM        NaCl, 10 mM sodium phosphate, 3% sucrose, pH7.3;    -   rat IgG1:20 mM sodium acetate, 7% sucrose pH 5.5; 11A11:20 mM        NaAc, 100 mM NaCl, 3% sucrose pH5.5; 11A11-G1:20 mM NaAc, 9%        sucrose, pH 5.5; 11A11-G2a: 20 mM NaAc, 9% sucrose, pH 5.5.

Tumor Cell Line Preparation and Implant:

CT26 colon carcinoma cells were cultured in RPMI medium supplementedwith 10% heat-inactivated fetal bovine serum. 3×10⁵ log-phase andsub-confluent CT26 cells were injected subcutaneously (SC) in a 100 μLvolume of serum-free RPMI in the right lower dorsal flank of each mouse.Mice were first shaved with electronic clippers in the area that wouldbe used for the implant.

CT-26 is a murine colorectal adenocarcinoma cell line syngeneic to theBALB/c mouse strain. CT-26 is a relevant model system for evaluating themechanism of action for an anti-PD-1 antibody because of thetranslatable molecular profile of this tumor post-anti-PD-1 therapy.

Tumor Measurements and Body Weights:

Tumors were measured the day before the first dose and twice a weekthereafter. Tumor length and width were measured using electroniccalipers and tumor volume determined using the formula Volume(mm³)=0.5×Length×Width² where length is the longer dimension. Mice wereweighed periodically to monitor general health but also to estimateactual mg/kg dose delivery per mouse where needed. Before treatment,mice were weighed and tumors from individual mice were measured. Toprevent bias, any outliers by weight or tumor volume were removed andthe remaining mice randomized into various treatment groups withequivalent mean tumor size. When the mean tumor volume in the CT26tumor-bearing mice reached ˜100 mm³, around 7 days post implant, animalswere randomized into treatment groups of 10 mice per group and dosingwas started. Animals were administered at the dosing concentrationsspecified below.

Dosing Solution Preparation, Administration, and Analyses:

Frozen stocks of the antibodies to be tested in the animal model werethawed and transferred to wet ice. To avoid repeated freeze thaw, eachvial of stock was thawed once and aliquots made in volumes sufficientfor one time use. Polypropylene, low adhesion tubes were used for thispurpose. The aliquots were snap frozen in dry ice and stored at −80° C.Before each dosing, one aliquot was thawed and diluted to nominalconcentration in the appropriate diluent and dosed immediately. Aliquotsof dosing solutions were snap frozen in dry ice and stored at −80° C.until analyses. Dosing solutions were assessed using the Meso ScaleDiscovery (MSD®, Rockville, Md.) platform which is based on multi-arraytechnology; a combination of electrochemiluminescence detection andpatterned arrays.

Dosing Anti-TIGIT/Anti-PD1 Treatment Results:

CT26 tumor-bearing BALB/cAnN mice were randomized into 10 treatmentgroups when the mean tumor volume of these mice reached a tumor sizeaverage of 100 mm³ (75 mm³-115 mm³): (1) muIgG1 isotype control+ ratIgG1 isotype control; (2) muIgG1 isotype control+ muIgG2a isotypecontrol; (3) muDX400+ rat IgG1 isotype control; (4) muDX400+ muIgG2aisotype control; (5) muIgG1 isotype control+11A11; (6) muIgG1 isotypecontrol+11A11-G1; (7) muIgG1 isotype control+11A11-G2a; (8)muDX400+11A11; (9) muDX400+11A11_G1; (10) muDX400+11A11_G2a. Animalswere administered rat anti-mouse TIGIT (11A11) or chimeric anti-TIGITantibodies 11A11-G1 or 11A11-G2a (described above) at a 20 mg/kg dose,IP, every 4 days for each of 6 cycles. An anti-mouse PD-1 (describedabove) was administered at a 10 mg/kg dose, IP, every 4 days for each of6 cycles. Post dosing, animals continued to be monitored and tumorvolumes were measured out to 54 days for some treatment groups. Tumorswere measured twice weekly. The results are shown in FIGS. 12 and 13.Single agent anti-TIGIT treatment using 11A11-G2a showed 52% tumorgrowth inhibition (TGI) at day 14; while little to no activity wasobserved using the 11A11 parental antibody or the 11A11-G1 antibody. Forsingle agent anti-PD1 treatment, 40-50% TGI was observed at day 14. Whencombined with isotype control rat IgG1, anti-PD-1 showed 10% completeregressions (CR) by day 28, such that 1 complete response out of 10animals was observed. The combination of anti-PD1 and parental 11A11showed 60% TGI at day 14 and demonstrated 30% CR by day 54. Thecombination of anti-PD1 and 11A11-G1 showed 56% TGI at day 14 anddemonstrated 30% CR by day 25. The combination of anti-PD1 and 11A11-G2ashowed 94% TGI at day 14 and demonstrated 70% CR at day 42. Thecombination of anti-PD1 and 11A11-G2a showed greater anti-tumor activityand more complete regressions compared to combinations of anti-PD1 with11A11 parental or 11A11-G1. There was no significant body weight lossassociated with administration of single agents or combination therapyindicating that treatments were well tolerated.

Example 9 Epitope Mapping of hTIGIT 14A6 Antibody by Hydrogen DeuteriumExchange Mass Spectrometry

The contact areas between anti-TIGIT antibody 14A6 and human TIGIT weredetermined by use of hydrogen deuterium exchange mass spectrometry(HDX-MS) analysis. HDX-MS measures the exchange of deuterium withhydrogen into the amide backbone of the protein. The exchange rate isinfluenced by the hydrogen's exposure to solvent. Comparison of theexchange levels in the antigen when the antibody is bound can identifyregions of the protein where the antibody is binding.

Materials

-   -   Human TIGIT-His—Comprising the extracellular domain of hTIGIT        (residues 25-150 of SEQ ID NO:31) and a histidine tag (SEQ ID        NO:87).    -   Rat anti-hTIGIT 14A6 antibody (comprising the VH/VL sequences of        SEQ ID NOs:7/8 (lot#78AGU) and a ratIgG2a Fc region)        (Rat×[TIGIT_H] mAb (LB155.14A6.G2.A8) IgG2a/Kappa (HY)).

Liquid Chromatography-Mass Spectrometry

The mass spectrometer was a Thermo Scientific Orbitrap-Velos. For themeasurement of deuterium labeled samples, the mass spectrometer was setto acquire one full scan MS data in the orbitrap at 60,000 resolvingpower, a target ion count of 1E6, a maximum ion injection time of 500milliseconds and two microscans. For the acquisition of MS/MS data forpeptide identifications, the mass spectrometer was set to acquire onefull scan spectrum at 60,000 resolving power followed by tendata-dependent MS/MS spectra in the ion trap.

The liquid chromatography system was a Waters® nanoACQUITY for theanalytical column gradient and a Waters® 515 isocratic pump for thesample digestion and loading. For sample digestion and loading, thebuffer used was 2% acetonitrile and 0.05% trifluoroacetic acid at a flowrate of 80 ul/min. For the analytical gradient, the buffers were BufferA) 0.1% formic acid in water and Buffer B) 0.1% formic acid inacetonitrile.

The gradient was at 40 ul/min from 2% B to 36% B in 10 minutes, followedby a wash of 80% B for 2 minutes and a reequilibration at 2% B for 3minutes. The column was then washed by cycling the gradient between 2%and 80% B, three times with 1 minute at each step, followed by a finalequilibration at 2% B for 5 minutes. The trapping column was a Waters®Vanguard C18 BEH 1.7 um Guard Column and the analytical column was aWaters® C18 BEH300, 1.7 um 1×50 mm column.

Sample handling for the deuterium labeling was done by a Leaptec H/D-XPAL system. The labeling sample tray was set to a temperature of 25° C.,the quenching tray was set to 1.5° C. and the trap and analytical columnchamber was set to 1.5° C. The immobilized pepsin column (PorosymeImmobilized Pepsin 2.1×30 mmm, Life Technologies) was kept outside thecolumn chamber at room temperature.

Deuterium Labeling

hTIGIT-His (30 pmol/μl) was mixed with an equal volume of the antibody(14 pmol/μl) or, in the unbound control, PBS pH 7.6. The antibody boundsamples and the unbound control were incubated at room temperature for 1hour before beginning the labeling experiment.

To deuterium label the samples, 2 μl of sample was mixed with 25 μl ofPBS in dueterium oxide pD 7.6. Labeling time points were 30, 300, 1500,4500 or 9000 seconds. After the set time, 25 μl of the labeling mixturewas added to 35 μl of cold quench buffer (8M Urea, 100 mM TCEP). Thequenched sample was incubated at 1.5° C. for one minute. 55 μl was theninjected into the column cooling chamber where the sample was passedover the pepsin column and the resulting peptides loaded onto thetrapping column. After three minutes, a valve switch took the pepsincolumn out of line and the trap was washed at additional one minute.After that the trap was switched inline with the analytical column andthe analytical gradient and the mass spectometer were started.

A fully deuterated sample was generated by incubating 2 μl of hTIGITwith 108 μl of deuterated denaturing buffer (4M Urea, 100 mM TCEP, 0.01%DDM in 99.5% deueterium oxide). The sample was incubated at roomtemperature overnight. 55 μl was then directly injected into the columnchamber and the data acquired as before.

Data Analysis

LC-MS/MS data was acquired of an unlabeled sample and searched beforedeuterium labeling to verify successful digestion of the proteins and togenerate a list of peptides from pepsin digestion. Data was databasesearched using Proteome Discoverer 1.4 and the SEQUEST HT searchalgorithm (ThermoFisher Scientific). The protein database used was thehuman TIGIT-His and anti-hTIGIT antibody sequences concatenated to theyeast Saccharomycese cerevisiae uniprot (5/20/13) database.

MS data from the deuterium labeling experience was processed byHDExaminer (version 1.3, Sierra Analytics). The mass and retention timeselected by the software for each peptide was verified manually.

Results

The human TIGIT peptides protected by bound 14A6 antibody areillustrated in the heatmap of FIG. 14 and correspond to amino acidresidues 54-57, 68-70 and 76-81 of SEQ ID NO:31. (These same amino acidsare shown as residues 30-33, 44-46 and 52-57 of SEQ ID NO:87.)

Example 10 Epitope Mapping of hTIGIT 31C6 Antibody by Hydrogen DeuteriumExchange Mass Spectrometry

The contact areas between anti-TIGIT antibody 31C6 and human TIGIT weredetermined by use of hydrogen deuterium exchange mass spectrometry(HDX-MS) analysis. HDX-MS measures the exchange of deuterium withhydrogen into the amide backbone of the protein. One factor influencingthe exchange rate is the hydrogen's exposure to solvent. Comparison ofthe exchange levels in the antigen when the antibody is bound canidentify regions of the protein where the antibody is binding.

Materials

-   -   Human TIGIT-His—Comprising the extracellular domain of hTIGIT        (residues 25-145 of SEQ ID NO:31) and a histidine tag (SEQ ID        NO:87).    -   Mouse anti-hTIGIT 31C6 antibody (lot#41AHK) (Mouse×[TIGIT_H] mAb        (MEB125.31C6.A1.205) IgG1/Kappa (HY))

Liquid Chromatography-Mass Spectrometry

The mass spectrometer was a Thermo Scientific Orbitrap-Elite. For themeasurement of deuterium labeled samples, the mass spectrometer was setto acquire one full scan MS data in the orbitrap at 120,000 resolvingpower, a target ion count of 1E6, a maximum ion injection time of 500milliseconds and two microscans. For the acquisition of MS/MS data forpeptide identifications, the mass spectrometer was set to acquire onefull scan spectrum at 120,000 resolving power followed by tendata-dependent MS/MS spectra in the ion trap.

The liquid chromatography system was a Waters nanoAcquity for theanalytical column gradient and a Waters 515 isocratic pump for thesample digestion and loading. For sample digestion and loading, thebuffer used was 2% acetonitrile and 0.05% trifluoroacetic acid at a flowrate of 80 ul/min. For the analytical gradient, the buffers were BufferA) 0.1% formic acid in water and Buffer B) 0.1% formic acid inacetonitrile.

The gradient was at 40 ul/min from 2% B to 36% B in 10 minutes, followedby a wash of 80% B for 2 minutes and a re-equilibration at 2% B for 3minutes. The column was then washed by cycling the gradient between 2%and 80% B, three times with 1 minute at each step, followed by a finalequilibration at 2% B for 5 minutes. The trapping column was a WatersVanguard CSH C18 1.7 um Guard Column and the analytical column was aWaters CSH C18, 1.7 um 1×50 mm column.

Sample handling for the deuterium labeling was done by a Leaptec H/D-XPAL system. The labeling sample tray was set to a temperature of 25° C.,the quenching tray was set to 1.5° C. and the trap and analytical columnchamber was set to 1.5° C. The immobilized pepsin column (Enzymate BEHPepsin, Waters corporation) was kept outside the column chamber at roomtemperature.

Deuterium Labeling

hTIGIT-His (63 pmol/ul) was mixed with an equal volume of the antibody(32 pmol/ul) or, in the unbound control, PBS pH 7.6. The antibody boundsamples and the unbound control were incubated at room temperature for 1hour before beginning the labeling experiment.

To deuterium label the samples, 2 μl of sample was mixed with 25 μl ofPBS in deuterium oxide pH 7.6. Labeling time points were 30, 300, 1500,4500, 9000 and 13500 seconds. After the set time, 25 μl of the labelingmixture was added to 35 μl of cold quench buffer (8M Urea, 100 mM TCEP).The quenched sample was incubated at 1.5° C. for one minute. 55 μl wasthen injected into the column cooling chamber where the sample waspassed over the pepsin column and the resulting peptides loaded onto thetrapping column. After three minutes, a valve switch took the pepsincolumn out of line and the trap was washed at additional one minute. Thetrap was then switched in-line with the analytical column and theanalytical gradient and the mass spectrometer data acquisition wasstarted. Each time point was acquired in triplicate in randomized order.

A fully deuterated sample was generated by incubating 2 μl of hTIGIT (63pmol/ul) with 108 μl of deuterated denaturing buffer (4M Urea, 100 mMTCEP, 0.01% DDM in 99.5% deuterium oxide). The sample was incubated atroom temperature overnight. 55 μl was then directly injected into thecolumn chamber and the data acquired as before.

Data Analysis

LC-MS/MS data was acquired of an unlabeled sample and database searchedto verify successful digestion of the proteins and to generate a list ofpeptides from the pepsin digestion. Database search was done usingProteome Discoverer 1.4 and the SEQUEST HT search algorithm(ThermoFisher Scientific). The protein database used was the humanTIGIT-His and anti-hTIGIT antibody sequences concatenated to the yeastSaccharomyces cerevisiae uniprot (5/20/13) database.

MS data from the deuterium labeling experience was processed byHDExaminer (version 1.3, Sierra Analytics). The mass and retention timeselected by the software for each peptide was verified manually.

Results

The human TIGIT peptides protected by bound 31C6 antibody areillustrated in the heatmap of FIG. 15 and correspond to amino acidresidues 53-57, 60-65, 68-70, 72-81, 94-95, 109-119 of SEQ ID NO:31.(These same amino acids are shown as residues 29-33, 36-41, 44-46,48-57, 70-71, 85-95 of SEQ ID NO:87.)

Example 11 In Vitro T-Cell Activity Assay for Humanized Anti-hTIGITAntibodies

We further analyzed the activity of various humanized variants of one ofthe antibodies of the invention (31C6). In one assay we characterizedthe functional consequence of blocking human TIGIT receptor usinghTIGIT-Jurkat cells. In this assay the hTIGIT-Jurkat were co-culturedwith JY cells engineered to express human CD155 (hCD155-JY). The JY cellline used is an Epstein-Barr virus (EBV) immortalized B celllymphoblastoid cell line. As in the assay described above in Example 3,when the hTIGIT Jurkat cells are stimulated with plate bound a-CD3 andco-cultured with parental JY cells (not expressing human CD155), theyproduce IL-2. However, when the hTIGIT—Jurkat are co-cultured withhCD155-JY, IL-2 levels were reduced in a ligand dependent manner.Treatment with anti-hTIGIT antibodies of the invention rescues IL-2production in this assay in a dose-dependent fashion.

In this assay 96-well flat-bottom plates were coated with mouseanti-human CD3 antibody (1 μm/ml in PBS; Clone HIT3a; BD PharmingenCat#555336) overnight at 4° C. The next day, hTIGIT expressing Jurkatcells (50,000) were plated in the pre-coated plates and pre-incubatedfor 30-60 minutes with mAb at varying concentrations. Human CD155expressing JY cells (50,000) were added to the culture. After incubationfor 18-24 h at 37° C. and 5.0% CO2, IL-2 levels were assessed in culturesupernatants by mesoscale (Human IL-2 Tissue Culture MESO Kit:Cat#K151AHB-2). The results are shown in FIG. 16. A titration of theparental mouse anti-hTIGIT clone 31C6 (MEB125.31C6.A1.205 mIgG1) from 30μg/ml down to 0.04 μg/ml gives an EC₅₀ of 0.730 μg/ml, and the sametitration of the mouse human 31C6 chimera (Mouse human chimeraMEB125.31C6.A1.205 IgG1) gives an EC50 of 0.910 μg/ml. (The mouse human31C6 chimera comprised the variable regions of the parental 31C6 clone(SEQ ID Nos: 94 and 95) and a human IgG1 region.) Similarly, a titrationof the 31C6 humanized variants from 30 μg/ml down to 0.04 μg/ml givesthe following EC₅₀s:

Humanized Variant EC₅₀ MEB125.31C6.A1.205 VH4/VL1 (An antibody 0.620μg/ml  comprising the VH of SEQ ID NO: 127 and VL of SEQ ID NO: 130, anda human IgG1 Fc region) MEB125.31C6.A1.205 VH5/VL4 (An antibody 1.2μg/ml comprising the VH of SEQ ID NO: 128 and VL of SEQ ID NO: 133, anda human IgG1 Fc region) MEB125.31C6.A1.205 VH5/VL3 (An antibody 1.2μg/ml comprising the VH of SEQ ID NO: 128 and VL of SEQ ID NO: 132, anda human IgG1 Fc region)

We also used a primary cell-based assay to demonstrate that thehumanized anti-hTIGIT antibodies had activity in primary cells. Severallots of human peripheral blood mononuclear cells (PBMCs) were screenedfor TIGIT expression after stimulation (mixed lymphocyte reactionstimulation and a-CD3 stimulation). PBMCs were then chosen for primarycell-based assays based on their TIGIT expression after stimulation.HuCD155-Fc was coated onto tissue culture plates and PBMCs werestimulated with anti-CD3.

In this assay 96-well high binding plates (Corning 3361) were coatedwith human CD155-Fc (in-house generated, 1 ug/ml in PBS) overnight at 4°C. The next day, 50,000 human PBMC (Precision Bioservice Cat#83000C-1.0,lot#12920 in RPMI+10% human serum Bio-world cat#30611043-1,lot#V15022401, RBCs were removed by BD Pharmlyse BD cat#555899) wereplated in the pre-coated plates and pre-incubated for 30-60 minutes withanti-TIGIT mAbs at varying concentrations. Anti-CD3 antibody(eBioscience 16-0037-85) at final concentration 1 ug/ml was then added.After incubation for 48 h at 37° C. and 5.0% CO2, Proinflammatorycytokines (IFNy, IL1β, IL6 and TNFα) were assessed in culturesupernatants by mesoscale assay (Human Proinflammaatory-4 I tissueculture MESO Kit: Cat#K15009B-4). As shown in FIG. 17, humanizedvariants of 31C6 were able to stimulate IL-6, TNFα and IFNγ in a dosedependent manner similar to the mouse human 31C6 chimeric antibody.

The labels in FIG. 17 correspond to the following antibodies:

-   -   Mouse human chimera MEB125.31C6.A1.205 IgG1 corresponds to an        antibody comprising the variable regions of the parental 31C6        clone (SEQ ID Nos: 94 and 95) and a human IgG1 region.)    -   MEB125.31C6.A1.205 VH4/VL1 corresponds to an antibody comprising        the VH of SEQ ID NO: 127 and VL of SEQ ID NO:130, and a human        IgG1 Fc region)    -   MEB125.31C6.A1.205 VH5/VL4 corresponds to an antibody comprising        the VH of SEQ ID NO: 128 and VL of SEQ ID NO:133, and a human        IgG1 Fc region)    -   MEB125.31C6.A1.205 VH5/VL3 corresponds to an antibody comprising        the VH of SEQ ID NO: 128 and VL of SEQ ID NO:133, and a human        IgG1 Fc region)

All references cited herein are incorporated by reference to the sameextent as if each individual publication, database entry (e.g. Genbanksequences or GeneID entries), patent application, or patent, wasspecifically and individually indicated to be incorporated by reference.This statement of incorporation by reference is intended by Applicants,pursuant to 37 C.F.R. §1.57(b)(1), to relate to each and everyindividual publication, database entry (e.g. Genbank sequences or GeneIDentries), patent application, or patent, each of which is clearlyidentified in compliance with 37 C.F.R. §1.57(b)(2), even if suchcitation is not immediately adjacent to a dedicated statement ofincorporation by reference. The inclusion of dedicated statements ofincorporation by reference, if any, within the specification does not inany way weaken this general statement of incorporation by reference.Citation of the references herein is not intended as an admission thatthe reference is pertinent prior art, nor does it constitute anyadmission as to the contents or date of these publications or documents.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

TABLE 4 Sequence Information SEQ ID Description NO: SEQUENCE 14A6 H-CDR11 SDYWG 14A6 H-CDR2 2 FITYSGSTSYNPSLKS 14A6 H-CDR3 3 MPSFITLASLSTWEGYFDF14A6 L-CDR1 4 KASQSIHKNLA 14A6 L-CDR2 5 YANSLQT 14A6 L-CDR3 6 QQYYSGWT14A6 PARENTAL 7 EVQLQESGPGLVKPSQSLSLTCSVTGSSIASDYWGWIRKFPGNKMEWMGFI VHTYSGSTSYNPSLKSRISITRDTSKNQFFLQLHSVTTDDTATYSCARMPSFITLASLSTWEGYFDFWGPGTMVTVSS 14A6 PARENTAL 8DIQMTQSPSLLSASVGDRVTLNCKASQSIHKNLAWYQQKLGEAPKFLIYYA VLNSLQTGIPSRFSGSGSGTDFTLTISGLQPEDVATYFCQQYYSGWTFGGGTK VELK Hul4A6VH.1 9EVQLQESGPGLVKPSETLSLTCTVSGGSISSDYWGWIRQPPGKGLEWIGFITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.1a 10EVQLQESGPGLVKPSETLSLTCTVSGGSISSDYWGWIRQPPGKGLEWIGFITYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.1b 11EVQLQESGPGLVKPSETLSLTCTVSGGSISSDYWGWIRQPPGKGLEWIGFITYSGSTSYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.1c 12EVQLQESGPGLVKPSETLSLTCTVSGSSISSDYWGWIRQPPGKGLEWMGFITYSGSTSYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.1d 13EVQLQESGPGLVKPSETLSLTCTVSGGSISSDYWGWIRQPPGKGLEWIGFITYSGSTSYNPSLKSRVTISRDTSKNQFSLKLHSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.1e 14EVQLQESGPGLVKPSETLSLTCTVSGGSISSDYWGWIRQPPGKGLEWIGFITYSGSTSYNPSLKSRITISRDTSKNQFSLKLHSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.1f 15EVQLQESGPGLVKPSETLSLTCTVSGSSISSDYWGWIRQPPGKGLEWIGFITYSGSTSYNPSLKSRITISRDTSKNQFSLKLHSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.1g 16EVQLQESGPGLVKPSETLSLTCTVSGSSISSDYWGWIRQPPGKGLEWMGFITYSGSTSYNPSLKSRITISVDTSKNQFSLKLHSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.2 17EVQLQESGPGLVKPSETLSLTCAVSGYSISSDYWGWIRQPPGKGLEWIGFITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.2a 18EVQLQESGPGLVKPSETLSLTCAVSGYSISSDYWGWIRQPPGKGLEWIGFITYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.2b 19EVQLQESGPGLVKPSETLSLTCAVSGYSISSDYWGWIRQPPGKGLEWIGFITYSGSTSYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.2c 20EVQLQESGPGLVKPSETLSLTCAVSGSSISSDYWGWIRQPPGKGLEWMGFITYSGSTSYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.2d 21EVQLQESGPGLVKPSETLSLTCAVSGYSISSDYWGWIRQPPGKGLEWIGFITYSGSTSYNPSLKSRVTISRDTSKNQFSLKLHSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.2e 22EVQLQESGPGLVKPSETLSLTCAVSGYSISSDYWGWIRQPPGKGLEWIGFITYSGSTSYNPSLKSRITISRDTSKNQFSLKLHSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.2f 23EVQLQESGPGLVKPSETLSLTCAVSGSSISSDYWGWIRQPPGKGLEWIGFITYSGSTSYNPSLKSRITISRDTSKNQFSLKLHSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6VH.2e 24EVQLQESGPGLVKPSETLSLTCAVSGSSISSDYWGWIRQPPGKGLEWMGFITYSGSTSYNPSLKSRITISRDTSKNQFSLKLHSVTAADTAVYYCARMPSFITLASLSTWEGYFDFWGQGTMVTVSS Hul4A6Vk.1 25DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKLLIYYANSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTK VEIK Hul4A6Vk.1a 26DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKFLIYYANSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTK VEIK Hul4A6Vk.1b 27DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKFLIYYANSLQTGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTK VEIK Hul4A6Vk.2 28DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKVPKLLIYYANSLQTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYYSGWTFGGGTK VEIK Hul4A6Vk.2a 29DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKVPKFLIYYANSLQTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYYSGWTFGGGTK VEIK Hul4A6Vk.2b 30DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKVPKFLIYYANSLQTGIPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYYSGWTFGGGTK VEIK Human TIGIT 31mrwcllliwa qglrqaplas gmmtgtiett gnisaekggs iilqchlsst taq vtqvnwe qqdqlla icn  adlgw hisps fkdrvapgpg lgltlqsltv ndtgeyfciy htypdgtytgriflevless vaehgarfqi pllgamaatl vvictavivvvaltrkkkal rihsvegdlr rksagqeews psapsppgscvqaeaapagl cgegrgedca elhdyfnvls yrslgncsff tetg Cyno/Rhesus 32mrwclfliwa qglrqaplas gmmtgtiett gnisakkggs TIGITvilqchlsst maqvtqvnwe qhdhsllair naelgwhiypafkdrvapgp glgltlqslt mndtgeyfct yhtypdgtyrgriflevles svaehsarfq ipllgamamm lvviciavivvvvlarkkks lrihsvesgl qrkstgqeeq ipsapsppgscvqaeaapag lcgeqqgddc aelhdyfnvl syrslgscsf ftetg Pembrolizumab 33QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGI Heavy chainNPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Pembrolizumab 34EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLL Light chainIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECNivolumab 35 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIheavy chain WYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Nivolumab 36EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDA light chainSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC16AHA_tigit_ 37 EVQLQESGPGLVKPSETLSLTCTVSGSSIASDYWGWIRQPPGKGLEWIGFI14a6_humanized_ TYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH1TLASLSTWEGYFDFWGQGTMVTVSSAS LB155.14A6.G2. A8_VH1 18AHA_tigit_ 38EVQLQESGPGLVKPSETLSLTCTVSGSSIASDYWGWIRQPPGKGLEWIGFI 14a6_humanized_TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH2TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH2 20AHA_tigit_ 39EVQLQESGPGLVKPSETLSLTCTVSGSSIASDYWGWIRKPPGKGLEWIGFI 14a6_humanized_TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH3TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH3 21AHA_tigit_ 40EVQLQESGPGLVKPSETLSLTCTVSGSSIASDYWGWIRQPPGKKLEWIGFI 14a6_humanized_TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH4TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH4 19AHA_tigit_ 41EVQLQESGPGLVKPSETLSLTCTVSGSSIASDYWGWIRQPPGKGMEWIGFI 14a6_humanized_TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH5TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH5 22AHA_tigit_ 42EVQLQESGPGLVKPSETLSLTCTVSGSSIASDYWGWIRKPPGKKMEWIGFI 14a6_humanized_TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH6TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH6 23AHA_tigit_ 43EVQLQESGPGLVKPSETLSLTCTVSGSSIASDYWGWIRQFPGKGLEWIGFI 14a6_humanized_TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTADDTAVYYCARMPSFI VH7TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH7 24AHA_tigit_ 44EVQLQESGPGLVKPSETLSLTCTVSGSSIASDYWGWIRKPPGKKMEWIGFI 14a6_humanized_TYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH8TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH8 25AHA_tigit_ 45EVQLQESGPGLVKPSETLSLTCSVTGSSIASDYWGWIRQPPGKGLEWIGFI 14a6_humanized_TYSGSTSYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH9TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH9 26AHA_tigit_ 46EVQLQQSGAGLLKPSETLSLTCSVTGSSIASDYWGWIRQPPGKGLEWIGFI 14a6_humanized_TYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH10TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH10 27AHA_tigit_ 47EVQLQESGPGLVKPPGTLSLTCSVTGSSIASDYWGWVRQPPGKGLEWIGFI 14a6_humanized_TYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMPSFI VH11TLASLSTWEGYFDFWGQGTMVTVSS LB155.14A6.G2. A8_VH11 09AHA_tigit_ 48DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKLLIYYA 14a6 humanized_NSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTK VL1 VEIKLB155.14A6.G2. A8_VL1 11AHA_tigit_ 49DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKFLIYYA 14a6 humanized_NSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTK VL2 VEIKLB155.14A6.G2. A8_VL2 12AHA_tigit_ 50DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKLLIYYA 14a6 humanized_NSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYYSGWTFGGGTK VL3 VEIKLB155.14A6.G2. A8_VL3 13AHA_tigit_ 51DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKFLIYYA 14a6 humanized_NSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYYSGWTFGGGTK VL4 VEIKLB155.14A6.G2. A8_VL4 15AHA_tigit_ 52DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKAPKLLIYYA 14a6 humanized_NSLQTGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSGWTFGGGTK VL5 VEIKLB155.14A6.G2. A8_VL5 Leader 53 MEWSWVFLFFLSVTTGVHS sequenceheavy chains Leader 54 MSVPTQVLGLLLLWLTDARC sequence light chainsHeavy chain 55 TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFconstant PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP domain-PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF IgG4NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK S228PGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGKKappa light 56 VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ chainESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR constant GEC domain28H5H-CDR1 57 GYSITSDYAWN 28H5H-CDR2 58 YISNSGSASYNPSLKS 28H5H-CDR3 59LIYYDYGGAMNF 28H5L-CDR1 60 KASQGVSTTVA 28H5L-CDR2 61 SASYRYT 28H5L-CDR362 QHYYSTPWT 28H5 PARENTAL 63DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGY VHISNSGSASYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCATLIYY DYGGAMNFWGQGTSVTVSS28H5 PARENTAL 64 DIVMTQSHKFMSTSVGDRVSITCKASQGVSTTVAWYQQKPGQSPKLLIYSA VLSYRYTGVPDRFTGSGSGTDFTFTISSVQSEDLAVYYCQHYYSTPWTFGGGT KLEIK 14H6 L-CDR2 65YASNLQT variant 14H6 L-CDR2 65 YASSLQT variant 14H6 L-CDR2 67 YASTLQTvariant 14H6 L-CDR2 68 YATTLQT variant 14H6 L-CDR2 69 YASYLQT variant14H6 L-CDR2 70 YANQLQT variant 14H6 L-CDR2 71 YAGSLQT variant14H6 L-CDR2 72 YASQLQT variant 14H6 L-CDR2 73 YADSLQT variant14H6 L-CDR3 74 QQYYSGFT variant 14H6 L-CDR3 75 QQYYSGYT variant14H6 L-CDR3 76 QQYYSGIT variant 14H6 L-CDR3 77 QQYYSGVT variant14H6 L-CDR3 78 QQYYSGLT variant 14H6 H-CDR3 79 MPSFITLASLSTFEGYFDFvariant 14H6 H-CDR3 80 MPSFITLASLSTYEGYFDF variant 14H6 H-CDR3 81MPSFITLASLSTIEGYFDF variant 14H6 H-CDR3 82 MPSFITLASLSTVEGYFDF variant14H6 H-CDR3 83 MPSFITLASLSTLEGYFDF variant Nucleic acid 84GATGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACencoding 28H5CTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGATCCGACAGTTTCPARENTAL VHCAGGAAACAAACTGGAGTGGATGGGCTACATAAGCAACAGTGGTAGCGCTAGCTACAACCCATCTCTCAAAAGTCGCATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAACCCTGATCTACTATGATTACGGGGGGGCTATGAACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA Nucleic acid 85GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATencoding 28H5CACCTGCAAGGCCAGTCAGGGTGTGAGTACTACTGTGGCCTGGTATCAACAGAAACCAGGACPARENTAL VLAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGTCTGAAGACCTGGCAGTTTATTACTGTCAGCATTATTATAGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA Heavy chain 86ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL constantYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF domain-LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV IgG1SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hTIGIT-HIS 87gtiett gnisaekggs iilqchlsst taqvtqvnwe qqdqllaicn adlgwhispsfkdrvapgpg lgltlqsltv ndtgeyfciy htypdgtytg riflevlessvaehgarfqi pllga hhhhhhhhhggq 31C6 H-CDR1 88 SYVMH 31C6 H-CDR2 89YIDPYNDGAKYNEKFKG 31C6 H-CDR3 90 GGPYGWYFDV 31C6 L-CDR1 91 RASEHIYSYLS31C6 L-CDR2 92 NAKTLAE 31C6 L-CDR3 93 QHHFGSPLT 31C6 PARENTAL 94EVQLQQSGPELVKPGSSVKMSCKASGYTFSSYVMHWVKQKPGQGLEWIGYIDPYNDGAKYNEVH (with CDRs KFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGPYGWYFDVWGAGTTVTVSSunderlined) 31C6 PARENTAL 95DIQMTQSPASLSASVGETVTITCRASEHIYSYLSWYQQKQGKSPQLLVYNAKTLAEGVPSRFVL (with CDRs SGSGSGTQFSLKINSLQPEDFGTYYCQHHFGSPLTFGAGTTLELK underlined)31C6 H-CDR2 96 YIDPYNrGAKYNEKFG VARIANT (D56R) 31C6 H-CDR2 97YIDPYNlGAKYNEKG VARIANT F (D56L) 31C6 H-CDR2 98 YIDPYNkGAKYNEKFG VARIANT(D56K) 31C6 H-CDR2 99 YIDPYNfGAKYNEKFG VARIANT (D56F) 31C6 H-CDR2 100YIDPYNsGAKYNEKFG VARIANT (D56S) 31C6 H-CDR2 101 YIDPYNyGAKYNEKFG VARIANT(D56Y) 31C6 H-CDR2 102 YIDPYNvGAKYNEKFG VARIANT (D56V) 31C6 H-CDR2 103YIDPYNDrAKYNEKFKG VARIANT (G57R) 31C6 H-CDR2 104 YIDPYNDnAKYNEKFKGVARIANT (G57N) 31C6 H-CDR2 105 YIDPYNDqAKYNEKFKG VARIANT (G57Q)31C6 H-CDR2 106 YIDPYNDeAKYNEKFKG VARIANT (G57E) 31C6 H-CDR2 107YIDPYNDlAKYNEKFKG VARIANT (G57L) 31C6 H-CDR2 108 YIDPYNDkAKYNEKFKGVARIANT (G57K) 31C6 H-CDR2 109 YIDPYNDsAKYNEKFKG VARIANT (G57S)31C6 H-CDR2 110 YIDPYNDyAKYNEKFKG VARIANT (G57Y) 31C6 H-CDR2 111YIDPYNDvAKYNEKFKG VARIANT (G57V) 31C6 L-CDR2 112 AAKTLAE variant (N50A)31C6 L-CDR2 113 YAKTLAE variant (N50Y) 31C6 L-CDR2 114 WAKTLAE variant(N50W) 31C6 L-CDR2 115 SAKTLAE variant (N50S) 31C6 L-CDR2 116 TAKTLAEvariant (N50T) 31C6 L-CDR2 117 IAKTLAE variant (N50I) 31C6 L-CDR2 118VAKTLAE variant (N50V) 31C6 L-CDR2 119 NNKTLAE variant (A51N)31C6 L-CDR2 120 NIKTLAE variant (A51I) 31C6 L-CDR2 121 NLLTLAE variant(A51L) 31C6 L-CDR2 122 NTKTLAE variant (A51T) 31C6 L-CDR2 123 NVKTLAEvariant (A51V) 31C6_HUMZ_VH1 124EVQLVQSGAEVKKPGASVKVSCKASGYTFSSYVMHWVRQAPGQRLEWIGYIDPYNDGAKYSQ(with CDRs KFQGRVTLTRDTSASTAYMELSSLRSEDTAVYYCARGGPYGWYFDVWGQGTTVTVSSunderlined) 31C6_HUMZ_VH2 125EVQLVQSGAEVKKPGASVKVSCKASGYTFSSYVMHWVRQAPGQRLEWIGYIDPYNDGAKYSQ(with CDRs KFQGRVTLTSDKSASTAYMELSSLRSEDTAVYYCARGGPYGWYFDVWGQGTTVTVSSunderlined) 31C6_HUMZ_VH3 126EVQLVQSGAEVKKPGASVKVSCKASGYTFSSYVMHWVRQAPGQGLEWIGYIDPYNDGAKYAQ(with CDRs KFQGRVTLTRDTSTSTVYMELSSLRSEDTAVYYCARGGPYGWYFDVWGQGTTVTVSSunderlined) 31C6_HUMZ_VH4 127EVQLVQSGAEVKKPGASVKVSCKASGYTFSSYVMHWVRQAPGQGLEWIGYIDPYNDGAKYAQ(with CDRs KFQGRVTLTSDKSTSTVYMELSSLRSEDTAVYYCARGGPYGWYFDVWGQGTTVTVSSunderlined) 31C6_HUMZ_VH5 128EVQLVQSGAEVKKPGSSVKVSCKASGYTFSSYVMHWVRQAPGQGLEWIGYIDPYNDGAKYAQ(with CDRs KFQGRVTLTSDKSTSTAYMELSSLRSEDTAVYYCARGGPYGWYFDVWGQGTTVTVSSunderlined) 31C6_HUMZ_VH6 129EVQLVQSGAEVKKPGASVKVSCKASGYTFSSYVMHWVRQAPGQGLEWIGYIDPYNDGAKYAQ(with CDRs KFQGRVTLTSDKSISTAYMELSRLRSDDTVVYYCARGGPYGWYFDVWGQGTTVTVSSunderlined) 31C6 Humz_L1 130DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKAPKLLIYNAKTLAE (with CDRsGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHFGSPLTFGQGTRLEIK underlined)31C6 Humz_L2 131DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKAPKLLIYNAKTLAE (with CDRsGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCQHHFGSPLTFGQGTRLEIK underlined)31C6 Humz_L3 132DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKVPKLLIYNAKTLAE (with CDRsGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHHFGSPLTFGQGTRLEIK underlined)31C6 Humz_L4 133DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKVPKLLIYNAKTLAE (with CDRsGVPSRFSGSGSGTQFTLTISSLQPEDVATYYCQHHFGSPLTFGQGTRLEIK underlined) 31C6 H-134 YIDPYNDGAKYAQKFQG CDR2 variant 31C6 H- 135 YIDPYNDGAKYSQKFQGCDR2 variant 18G10-VH 136QVQLMESGPGLVQPSQTLSLTCTVSGFPLTSYTVHWVRQPPGKGLEWIGAIWSSGSTDYNSA sequenceLKSRLNINRDSSKSQVFLKMNSLQTEDTAIYFCTKSGWAFFDYWGQGVMVTVSS 18G10-VL 137DIQMTQSPSLLSASVGDRVTLNCIASQNIYKSLAWYQLKLGEAPKLLIYNANSLQAGIPSRF sequenceSGSGSGTDFALTISGLQPEDVATYFCQQYSGGYTFGAGTKLELK 11A11-VH 138EVQLVESGGDLVQPGRSLKISCVASGFTFSDYYMAWVRLAPQKGLEWVASISYEGSRTHYGD sequenceSVRGRFIISRDNPKNILYLQMNSLGSEDTATYFCARHTGTLDWLVYWGQGTLVIVSS 11A11-VL 139NIVMAQSPKSMSISAGDRVTMNCKASQNVDNNIAWYQQKPGQSPKLLIFYASNRYSGVPDRF sequenceTGGGYGTDFTLTIKSVQAEDAAFYYCQRIYNFPTFGSGTKLEIK 14A6 H-CDR3 140MPSFITLASLSTXEGYFDF CONSENSUS X= W, F, Y, I, V, L 14A6 L-CDR2 141YAX₁X₂LQT CONSENSUS X₁ = N, S, T, G, D X₂ = S, N, S, T, Y, Q 14A6 L-CDR3142 QQYYSGXT CONSENSUS X = W, F, Y, I, V, L 14A6 VH 143EVQLQX₁SGX₂GLX₃KPX₄X₅X₆LSLTCX₇VX₈GX₃₀SIX₃₁ SDYWGWX₉RX₁₀X₁₁PGX₁₂X₁₃X₁₄EWPARENTALX₁₅GFITYSGSTSYNPSLKSRX₁₆X₁₇IX₁₈X₁₉DTSKNQFX₂₀LX₂₁LX₂₂SVTX₂₃X₂₄DTAX₂₅YCONSENSUS X₂₆CARMPSFITLASLSTX ₂₇ EGYFDFWGX₃₂GTX₂₈X₂₉TVSS X₁ = E or QX₂ = P or A X₃ = V or L X₄ = S or P X₅ = 4 or E or G X₆ = S or T X₇ =S or T or A X₈ = T or S X₉ = I or V X₁₀ = K or Q X₁₁ = F or P X₁₂ =N or K X₁₃ = K or G X₁₄ = M Or L X₁₅ = M Or I X₁₆ = I or V X₁₇ = S or TX₁₈ = T or S X₁₉ = R or V X₂₀ = F or S X₂₁ = Q or K X₂₂ = H or S X₂₃ =T or A X₂₄ = D or A X₂₅ = T or V X₂₆ = S or Y, X₂₇ = W, F, Y, I, V or LX₂₈ = N, V, L, A, R, N, P, Q, E, G, I, H, K, F, S, T, W or Y X₂₉ =V, T or L X₃₀ = S or G or Y X₃₁ = A or S X₃₁ = P or Q 14A6 VH 144EVQLQX₁SGX₂GLX₃KPX₄X₅TLSLTCX₆VX₇GX₈SIX₉SDYWGWX₁₀RX₁₁X₁₂PGKX₁₃X₁₄EWX₁₅HUMANIZEDGFITYSGSTSYNPSLKSRX₁₆TISX₁₇DTSKNQFSLKLX₁₈SVTAX₁₉DTAVYYCARMPSFITLACONSENSUS SLSTX ₂₀ EGYFDFWGQGTX₂₁X₂₂TVSS X₁ = E or Q X₂ = P or A X₃ =V or L X₄ = S or P X₅ = E or G X₆ = T or A or S X₇ = S or T X₈ =G or S or Y X₉ = S or A X₁₀ = I Or V X₁₁ = Q or K X₁₂ = P or F X₁₃ =G or K X₁₄ = L Or M X₁₅ = I Or M X₁₆ = V Or I X₁₇ = V Or R X₁₈ = S or HX₁₉ = A or D X₂₀ = W, F, Y, I, V, L X₂₁ =M, V, L, A, R, N, P, Q, E, G, I, H, K, F, S, T, W or Y X₂₂ = V, T or L14A6 VL 145 DIQMTQSPSX₁LSASVGDRVTX₂X₃CKASQSIHKNLAWYQQKX₄GX₅X₁₅PKX₆LIYYAX₇ X ₈ LQT PARENTAL GX₉PSRFSGSGSGTDFTLTISX₁₀LQPEDX₁₁ATYX₁₂CQQYYSGX ₁₃TFGGGTKVEX₁₄K CONSENSUS X₁ = L or S X₂ = L or I X₃ = N or T X₄ = L or PX₅ = E or K X₆ = F or L X₇ = N, S, T, G or D X₈ = S, N, T, Y or Q X₉ =I or V X¹⁰ = G or S X¹¹ = V or F X¹² = F or Y X¹³ = W, F, Y, I, V or LX¹⁴ = L Or I X¹⁵ = A or V 14A6 VL 146DIQMTQSPSSLSASVGDRVTITCKASQSIHKNLAWYQQKPGKX₆PKX₁LIYYAX ₂ X ₃ LQTGX₄PHUMANIZED SRFSGSGSGTDFTLTISSLQPEDX₇ATYYCQQYYSGX ₅ TFGGGTKVEIK CONSENSUSX₁ = L or F X₂ = N, S, T, G or D X₃ = S, N, T, Y or Q X₄ = V or I X₅ =W, F, Y, I, V or L X₆ = A or V X₇ = F or V 31C6 H-CDR2 147YIDPYNX₁X₂AKYX₃X₄KFX₅G CONSENSUS X₁ = D, R, L, K, F, S, Y or V X₂ =G, R, N, Q, E, L K, S, Y or V X₃ = N, A or S X₄ = E or Q X₅ = K or Q31C6 L-CDR2 148 X₁X₂KTLAE CONSENSUS X₁ =N, A, V, W, S, T, R, H G, I or VX₂ =A, N, I, L, T or V 31C6 VH 149EVQLX₁QSGX₂EX₃X₄KPGX₅SVKX₆SCKASGYTFSSYVMHWVX₇QX₈PGQX₉LEWIGYIDPYNPARENTAL X ₁₀ X ₁₁ AKYX ₁₂ X ₁₃ KFX ₁₄GX₁₅X₁₆TLTX₁₇DX₁₈SX₁₉STX₂₀YMELSX₂₁LX₂₂SX₂₃DX₂₄X₂₅VYYC CONSENSUSARGGPYGX₂₆ YFDVWGX₂₇GTTVTVSS X₁ = Q or V X₂ = P or A X₃ = V or L X₄ =V or K X₅ = S or A X₆ = M or V X₇ = K or R X₈ = K or A X₉ = G or R X₁₀ =D, R, L, K, F, S, Y or V X₁₁ = G, R, N, Q, E, L K, S, Y or V X₁₂ =N, A or S X₁₃ = E or Q X₁₄ = K or Q X₁₅ = R or K X₁₆ = A or V X₁₇ =S Or R X₁₈ = K or T X₁₉ = S, I, A or T X₂₀ = A or V X₂₁ = R or S X₂₂ =T Or R X₂₃ = D Or E X₂₄ = S Or T X₂₅ = A or V X₂₆ =W, A, D, E, F, G, I, K, N, Q, R, S, T, V or Y X₂₇ = A Or Q 31C6 VH 150EVQLVQSGAEVKKPGX₁SVKVSCKASGYTFSSYVMHWVRQAPGQX₂LEWIG HUMANIZED YIDPYNX ₃X ₄ AKYX ₅ X ₅ KFX ₇ GRVTLTX₈DX₉SX₁₀STX₁₁YMELSX₁₂LRSX₁₃DT CONSENSUSX₁₄VYYCARGGPYGX ₁₅ YFDVWGQGTTVTVSS X₁ = A or S X₂ = R or G X₃ =D, R, L, K, F, S, Y or V X₄ = G, R, N, Q, E, L K, S, Y or V X₅ =N, A or S X₆ = E or Q X₇ = K or Q X₈ = R or S X₉ = T or K X₁₀ =A, T or I X₁₁ = A or V X₁₂ = S Or R X₁₃ = E or D X₁₄ = A or V X₁₅ =W, A, D, E, F, G, I, K, N, Q, R, S, T, V or Y 31C6 VL 151DIQMTQSPX₁SLSASVGX₂X₃VTITCRASEHIYSYLSWYQQKX₄GKX₅PX₆LLX₇YX ₈ X ₉ KTLAEPARENTALGVPSRFSGSGSGTX₁₀FX₁₁LX₁₂IX₁₃SLQPEDX₁₄X₁₅TYYCQHHFGSPLTFGX₁₆GTX₁₇LEX₁₈CONSENSUS K X₁ = A or S X₂ = E or D X₃ = T or R X₄ = Q or P X₅ =S, A or V X₆ = Q or K X₇ = V or I X₈ = N, A, Y, W, S, T, I or V X₉ =A, N, I, L, T or V X₁₀ = Q or D X₁₁ = S or T X₁₂ = K or T X₁₃ = N or SX₁₄ = F or V X₁₅ = G or A X₁₆ = A or Q X₁₇ = T or R X₁₈ = L or I31C6 L-VL 152 DIQMTQSPSSLSASVGDRVTITCRASEHIYSYLSWYQQKPGKX₁PKLLIYHUMANIZED X ₂ X ₃ KTLAEGVPSRFSGSGSGTX₄FTLTISSLQPEDX₅ATYYCQHHFGSPLTFGQGTRCONSENSUS LEIK X₁ = A or V X₂ = N, A, Y, W, S, T, I or V X₃ =A, N, I, L, T or V X₄ = D or Q X₅ = F or V 31C6 H-CDR3 153 GGPYGXYFDVCONSENSUS X₁₅ = W, A, D, E, F, G, I, K, N, Q, R, S, T, V or Y31C6 H-CDR3 154 GGPYGAYFDV VARIANT 31C6 H-CDR3 155 GGPYGDYFDV VARIANT31C6 H-CDR3 156 GGPYGEYFDV VARIANT 31C6 H-CDR3 157 GGPYGFYFDV VARIANT31C6 H-CDR3 158 GGPYGGYFDV VARIANT 31C6 H-CDR3 159 GGPYGIYFDV VARIANT31C6 H-CDR3 160 GGPYGKYFDV VARIANT 31C6 H-CDR3 161 GGPYGNYFDV VARIANT31C6 H-CDR3 162 GGPYGQYFDV VARIANT 163 GGPYGRYFDV 31C6 H-CDR3 164GGPYGSYFDV VARIANT 165 GGPYGTYFDV 31C6 H-CDR3 166 GGPYGVYFDV VARIANT 167GGPYGYYFDV

1. An antibody or antigen binding fragment thereof that binds to humanTIGIT, wherein the antibody or antigen binding fragment is selected fromthe group consisting of: a. an antibody or antigen binding fragmentcomprising: a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO:1, a heavy chain variable region CDR2 comprisingthe amino acid sequence of SEQ ID NO:2, a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO:3, 79, 80, 81, 82,83 or 140, a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO:4, a light chain variable region CDR2 comprisingthe amino acid sequence of SEQ ID NO:5, 65, 66, 67, 68, 69, 70, 71, 72,73 or 41, and a light chain variable region CDR3 comprising the aminoacid sequence of SEQ ID NO:6, 74, 75, 76, 77, 78 or 142; b. an antibodyor antigen binding fragment comprising: a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO:57, a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:58,a heavy chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO:59, a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO:60, a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO:61, and a light chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO:62;and c. an antibody or antigen binding fragment comprising: a heavy chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:88,a heavy chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO:89, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 134, 135 or 147, a heavy chain variable region CDR3comprising the amino acid sequence of SEQ ID NO:90, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166 or 167, a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:91,a light chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO:92, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 or148, and a light chain variable region CDR3 comprising the amino acidsequence of SEQ ID NO:93.
 2. The antibody or antigen binding fragment ofclaim 1, wherein the antibody comprises a heavy chain variable regionCDR1 comprising the amino acid sequence of SEQ ID NO:1, a heavy chainvariable region CDR2 comprising the amino acid sequence of SEQ ID NO:2,a heavy chain variable region CDR3 comprising the amino acid sequence ofSEQ ID NO:3, a light chain variable region CDR1 comprising the aminoacid sequence of SEQ ID NO:4, a light chain variable region CDR2comprising the amino acid sequence of SEQ ID NO:5, and a light chainvariable region CDR3 comprising the amino acid sequence of SEQ ID NO:6.3. The antibody or antigen binding fragment of claim 1, wherein theantibody comprises a heavy chain variable region CDR1 comprising theamino acid sequence of SEQ ID NO:88, a heavy chain variable region CDR2comprising the amino acid sequence of SEQ ID NO:89, 134 or 135, a heavychain variable region CDR3 comprising the amino acid sequence of SEQ IDNO:90, a light chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO:91, a light chain variable region CDR2 comprisingthe amino acid sequence of SEQ ID NO:92, and a light chain variableregion CDR3 comprising the amino acid sequence of SEQ ID NO:93.
 4. Theantibody or antigen binding fragment of claim 1, wherein the antibodycomprises a heavy chain variable region CDR1 comprising the amino acidsequence of SEQ ID NO:88, a heavy chain variable region CDR2 comprisingthe amino acid sequence of SEQ ID NO: 134, a heavy chain variable regionCDR3 comprising the amino acid sequence of SEQ ID NO:90, a light chainvariable region CDR1 comprising the amino acid sequence of SEQ ID NO:91,a light chain variable region CDR2 comprising the amino acid sequence ofSEQ ID NO:92, and a light chain variable region CDR3 comprising theamino acid sequence of SEQ ID NO:93.
 5. An antibody or antigen bindingfragment thereof that binds to human TIGIT comprising a light chainimmunoglobulin, a heavy chain immunoglobulin or both a light chain and aheavy chain immunoglobulin selected from the group consisting of: a. anantibody or antigen binding fragment thereof comprising a variable heavychain comprising the amino acid sequence of SEQ ID NO:7 and/or avariable light chain comprising the amino acid sequence of SEQ ID NO:8;b. an antibody or antigen binding fragment thereof comprising a variableheavy chain comprising the amino acid sequence of SEQ ID NO:63 and/or avariable light chain comprising the amino acid sequence of SEQ ID NO:64;c. an antibody or antigen binding fragment thereof comprising a variableheavy chain comprising the amino acid sequence of SEQ ID NO: 94 and/or avariable light chain comprising the amino acid sequence of SEQ ID NO:95;d. an antibody or antigen binding fragment thereof comprising a variableheavy chain selected from the group consisting of SEQ ID NOs: 9-24,37-47 and 63 and/or a variable light chain selected from the groupconsisting of any one of SEQ ID NOs: 25-30, 48-52 and 64; e. an antibodyor antigen binding fragment thereof comprising a variable heavy chainselected from the group consisting of SEQ ID NOs: 124-129 and/or avariable light chain selected from the group consisting of any one ofSEQ ID NOs: 130-133; f. an antibody or antigen binding fragment thereofcomprising a variable heavy chain comprising the amino acid sequence ofSEQ ID NO:128 and/or a variable light chain comprising the amino acidsequence of SEQ ID NO:132; g. an antibody or antigen binding fragmentthereof comprising a variable heavy chain comprising the amino acidsequence of SEQ ID NO:127 and/or a variable light chain comprising theamino acid sequence of SEQ ID NO:130; and h. an antibody or antigenbinding fragment thereof comprising a variable heavy chain comprisingthe amino acid sequence of SEQ ID NO:128 and/or a variable light chaincomprising the amino acid sequence of SEQ ID NO:133; i. an antibody orantigen binding fragment thereof comprising a variable heavy chaincomprising the amino acid sequence of SEQ ID NO:143 and/or a variablelight chain comprising the amino acid sequence of SEQ ID NO:145; j. anantibody or antigen binding fragment thereof comprising a variable heavychain comprising the amino acid sequence of SEQ ID NO:149 and/or avariable light chain comprising the amino acid sequence of SEQ IDNO:151; k. an antibody or antigen binding fragment thereof comprising avariable heavy chain comprising the amino acid sequence of SEQ ID NO:144and/or a variable light chain comprising the amino acid sequence of SEQID NO:146; l. an antibody or antigen binding fragment thereof comprisinga variable heavy chain comprising the amino acid sequence of SEQ IDNO:150 and/or a variable light chain comprising the amino acid sequenceof SEQ ID NO:152; m. an antibody or antigen binding fragment thereofcomprising a variable heavy chain comprising at least 90%, 95%, 96%,97%, 98% or 99% identity any one of SEQ ID NOs: 7, 9-24, 37-47 and 63and/or a variable light chain comprising at least 90%, 95%, 96%, 97%,98% or 99% identity to any one of SEQ ID NOs: 8, 25-30, 48-52 and 64; n.an antibody or antigen binding fragment thereof comprising a variableheavy chain comprising at least 90%, 95%, 96%, 97%, 98% or 99% identityany one of SEQ ID NOs: 7, 9-24, 37-47 and 63 and/or a variable lightchain comprising at least 90%, 95%, 95%, 96%, 97%, 98% or 99% identityto any one of SEQ ID NOs: 8, 25-30, 48-52 and 64, wherein any sequencevariations occur in the framework regions of the antibody; o. anantibody or antigen binding fragment thereof comprising a variable heavychain comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity anyone of SEQ ID NOs: 124-129 and/or a variable light chain comprising atleast 90%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs:130-133; and p. an antibody or antigen binding fragment thereofcomprising a variable heavy chain comprising at least 90%, 95%, 96%,97%, 98% or 99% identity any one of SEQ ID NOs: 124-129 and/or avariable light chain comprising at least 90%, 95%, 95%, 96%, 97%, 98% or99% identity to any one of SEQ ID NOs: 130-133, wherein any sequencevariations occur in the framework regions of the antibody; q. anantibody or antigen binding fragment thereof comprising a variable heavychain comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity SEQ IDNO:128 and/or a variable light chain comprising at least 90%, 95%, 96%,97%, 98% or 99% identity to any one of SEQ ID NOs: 132; r. an antibodyor antigen binding fragment thereof comprising a variable heavy chaincomprising at least 90%, 95%, 96%, 97%, 98% or 99% identity SEQ IDNO:128 and/or a variable light chain comprising at least 90%, 95%, 96%,97%, 98% or 99% identity to any one of SEQ ID NOs: 132, wherein anysequence variations occur in the framework regions of the antibody; s.an antibody or antigen binding fragment thereof comprising a variableheavy chain comprising at least 90%, 95%, 96%, 97%, 98% or 99% identitySEQ ID NO:127 and/or a variable light chain comprising at least 90%,95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs: 130; t. anantibody or antigen binding fragment thereof comprising a variable heavychain comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity SEQ IDNO:127 and/or a variable light chain comprising at least 90%, 95%, 96%,97%, 98% or 99% identity to any one of SEQ ID NOs: 130, wherein anysequence variations occur in the framework regions of the antibody; u.an antibody or antigen binding fragment thereof comprising a variableheavy chain comprising at least 90%, 95%, 96%, 97%, 98% or 99% identitySEQ ID NO:128 and/or a variable light chain comprising at least 90%,95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs: 133; v. anantibody or antigen binding fragment thereof comprising a variable heavychain comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity SEQ IDNO:128 and/or a variable light chain comprising at least 90%, 95%, 96%,97%, 98% or 99% identity to any one of SEQ ID NOs: 133, wherein anysequence variations occur in the framework regions of the antibody. 6.The antibody or antigen binding fragment of claim 1, wherein theantibody or antigen binding fragment thereof binds to human TIGIT with aKD value of about 1×10-9 M to about 1×10-12 M as determined by surfaceplasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa orOCTET).
 7. An antibody or antigen binding fragment thereof that binds tothe same epitope of human TIGIT as an antibody selected from the groupconsisting of: a. an antibody comprising the variable heavy chain of SEQID NO:7 and the variable light chain of SEQ ID NO:8, b. an antibodycomprising the variable heavy chain of SEQ ID NO:63 and the variablelight chain of SEQ ID NO:64, and c. an antibody comprising the variableheavy chain of SEQ ID NO:94 and the variable light chain of SEQ IDNO:95; wherein the antibody or fragment thereof has at least one of thefollowing characteristics: i. binds to human TIGIT with a KD value ofabout 1×10-9 M to about 1×10-12 M as determined by surface plasmonresonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET);ii. cross-reacts with cynomolgous and rhesus TIGIT; iii. blocks bindingof human TIGIT to human CD155 and human CD112; iv. increases T cellactivation; v. stimulates antigen-specific T-cell production of IL-2 andIFNγ; vi. blocks induction of T cell suppression of activation inducedby TIGIT ligation with cognate ligands CD155 and CD112.
 8. The antibodyor antigen binding fragment of claim 7, wherein the antibody binds to anepitope comprising the following regions of human TIGIT (SEQ ID NO:31):residues 54-57, 68-70 and 76-81.
 9. The antibody or antigen bindingfragment of claim 7, wherein the antibody binds to an epitope comprisingthe following regions of human TIGIT (SEQ ID NO:31): residues 53-57,60-65, 68-70, 72-81, 94-95, and 109-119.
 10. The antibody or antigenbinding fragment of claim 1, which is a humanized antibody comprisingtwo heavy chains and two light chains.
 11. The antibody or antigenbinding fragment of claim 1, which is a humanized antibody, wherein theantibody comprises a human IgG1 constant domain and a human kappaconstant domain.
 12. (canceled)
 13. An isolated polypeptide comprisingthe amino acid sequence of any one of SEQ ID NOs: 7-30, 37-52, 63-64,94-95 or 124-133, 136-139, 143-146, 149-151.
 14. An isolated nucleicacid encoding: any one of the antibodies or antigen binding fragments ofclaim
 1. 15. An expression vector comprising the isolated nucleic acidof claim
 14. 16. A host cell comprising the expression vector of claim15.
 17. (canceled)
 18. A composition comprising the antibody or antigenbinding fragment of claim 1 and a pharmaceutically acceptable carrier ordiluent.
 19. The composition of claim 18, further comprising an agentselected from the group consisting of: a. an anti-PD1 antibody or anantigen binding fragment thereof; b. an anti-LAG3 antibody or an antigenbinding fragment thereof; c. an anti-VISTA antibody or an antigenbinding fragment thereof; d. an anti-BTLA antibody or an antigen bindingfragment thereof; e. an anti-TIM3 antibody or an antigen bindingfragment thereof; f. an anti-CTLA4 antibody or an antigen bindingfragment thereof; g. an anti-HVEM antibody or an antigen bindingfragment thereof; h. an anti-CD27 antibody or an antigen bindingfragment thereof; i. an anti-CD137 antibody or an antigen bindingfragment thereof; j. an anti-OX40 antibody or an antigen bindingfragment thereof; k. an anti-CD28 antibody or an antigen bindingfragment thereof; l. an anti-PDL1 antibody or an antigen bindingfragment thereof; m. an anti-PDL2 antibody or an antigen bindingfragment thereof; n. an anti-GITR antibody or an antigen bindingfragment thereof; o. an anti-ICOS antibody or an antigen bindingfragment thereof; p. an anti-SIRPα antibody or an antigen bindingfragment thereof; q. an anti-ILT2 antibody or an antigen bindingfragment thereof; r. an anti-ILT3 antibody or an antigen bindingfragment thereof; s. an anti-ILT4 antibody or an antigen bindingfragment thereof; and t. an anti-ILT5 antibody or an antigen bindingfragment thereof.
 20. The composition of claim 19, wherein the anti-PD1antibody or an antigen binding fragment thereof is selected from thegroup consisting of: pembrolizumab or an antigen binding fragmentthereof and nivolumab or an antigen binding fragment thereof. 21.(canceled)
 22. A method of treating cancer in a human subject,comprising administering to the subject an effective amount of theantibody or antigen binding fragment of claim 1, optionally inassociation with a further therapeutic agent or therapeutic procedure.23. A method of treating an infection or infectious disease in a humansubject, comprising administering to the subject an effective amount ofthe antibody or antigen binding fragment of claim 1, optionally inassociation with a further therapeutic agent or therapeutic procedure.24. A vaccine comprising the antibody or antigen binding fragment ofclaim 1 and an antigen.
 25. (canceled)
 26. (canceled)
 27. (canceled) 28.(canceled)
 29. (canceled)
 30. (canceled)
 31. (canceled)
 32. (canceled)33. (canceled)
 34. (canceled)